Nucleic acid vaccines for coronavirus

ABSTRACT

Provided herein are therapeutic nucleic acid molecules for managing, preventing and/or treating infectious diseases caused by coronavirus. Also provided herein are therapeutic compositions, including vaccines and lipid nanoparticles, comprising the therapeutic nucleic acids and related therapeutic methods and uses.

1. CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to Chinese PatentApplication No.: 202010276288.0 filed on Apr. 9, 2020, U.S. ProvisionalApplication No. 63/011,116 filed on Apr. 16, 2020, and Chinese PatentApplication No.: 202110293284.8 filed on Mar. 19, 2021, the contents ofeach of which is incorporated by reference in its entirety.

2. FIELD

The present disclosure generally relates to nucleic acid molecules thatcan be used for the management, prevention, and treatment of coronavirusinfection. The present disclosure also relates to lipid-containingcompositions, including vaccines, of the nucleic acid molecules, andrelated methods of delivery.

3. BACKGROUND

Coronaviruses pose serious health threats to humans and other animals.From 2002 to 2003, severe acute respiratory syndrome coronavirus(SARS-CoV) infected 8,000 people, with a fatality rate of ˜9%. Since2012, Middle East respiratory syndrome coronavirus (MERS-CoV) hasinfected more than 1,700 people, with a fatality rate of ˜36%. Since2013, porcine epidemic diarrhea coronavirus (PEDV) has swept throughoutthe United States, causing an almost 100% fatality rate in piglets andwiping out more than 10% of America's pig population in less than ayear. In March 2020, the World Health Organization (WHO) announced apandemic caused by the outbreak of Coronavirus Disease 2019 (COVID-19),which swept into more than 180 countries and killed more than 80,000people in the first few months of the outbreak. In general, the diseaseis caused by a newly discovered coronavirus, SARS-CoV-2, which showssymptoms of widespread respiratory, gastrointestinal, and centralnervous system diseases in humans and other animals, threatening humanhealth and causing economic loss. Therefore, there exist an urgent needfor effective therapeutics, including vaccines for curbing coronavirusinfections. The present disclosure meets this need.

4. SUMMARY

In one aspect, provided herein are non-naturally occurring nucleic acidmolecules that can be used for the prevention, management and treatmentof infectious diseases. In some embodiments, the non-naturally occurringnucleic acids encode a viral peptide or protein derived from coronavirusSARS-CoV-2. In some embodiments, the non-naturally occurring nucleicacid encode a viral peptide or protein derived from a coronaviruscomprising a genome, wherein the genome comprises the nucleic acidsequence set forth in SEQ ID NO:1.

In some embodiments, the non-naturally occurring nucleic acid moleculecomprises a coding region, wherein the coding region comprises one ormore open reading frames (ORFs), and wherein at least one ORFs encodesthe viral peptide or protein. In some embodiments, at least one ORFsencodes a heterologous peptide or polypeptide. In some embodiments, theheterologous peptide or polypeptide is an immuno-stimulating peptide orprotein. In some embodiments, the ORF encodes a fusion proteincomprising the viral peptide or protein fused to a heterologous peptideor polypeptide. In some embodiments, the heterologous peptide orpolypeptide is selected from a Fc region of human immunoglobulin, asignal peptide, and a peptide facilitating multimerization of the fusionprotein.

In some embodiments, the one or more ORFs consists a coding sequence asset forth in Tables 1 to 4. In some embodiments, the one or more ORFsconsists a coding sequence selected from SEQ ID NOS:3, 5, 7, 9, 11, 13,15, 17, 19, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45, or a transcribedRNA sequence thereof. In some embodiments, the one or more ORFs encodesa peptide or protein selected from SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 1618, 20-26, 28, 30, 32, 34, 36, 38, 40, 42, and 44.

In some embodiments, the non-naturally occurring nucleic acid moleculefurther comprises a 5′ untranslated region (5′-UTR), wherein the 5′-UTRcomprises the sequence set forth in SEQ ID NOS: 46-51. In someembodiments, the non-naturally occurring nucleic acid molecule furthercomprises a 3′ untranslated region (3′-UTR), wherein the 3′-UTRcomprises the sequence set forth in SEQ ID NOS: 52-57. In someembodiments, the 3′-UTR further comprises a poly-A tail or apolyadenylation signal.

In some embodiments, the non-naturally occurring nucleic acid moleculefurther comprises one or more functional nucleotide analogs that areselected from pseudouridine, 1-methyl-pseudouridine and5-methylcytosine. In some embodiments, the non-naturally occurringnucleic acid molecule further comprises the nucleic acid is DNA or mRNA.

In some embodiments, disclosed herein are vectors or cells comprisingthe naturally occurring nucleic acid molecule as described herein. Insome embodiments, disclosed herein are compositions comprising thenaturally occurring nucleic acid molecule as described herein. In someembodiments, the composition is formulated as lipid nanoparticlesencapsulating the nucleic acid in a lipid shell. In some embodiments,the composition is a pharmaceutical composition.

In one aspect, provided herein are pharmaceutical compositionscomprising at least one nucleic acid encoding a viral peptide or proteinderived from coronavirus SARS-CoV-2. In some embodiments, providedherein are pharmaceutical compositions comprising at least one nucleicacid encoding a viral peptide or protein derived from a coronaviruscomprising a genome, wherein the genome comprises the nucleic acidsequence set forth in SEQ ID NO:1.

In some embodiments of the pharmaceutical composition described herein,the viral peptide or protein is selected from: (a) a spike (S) proteinof the coronavirus, (b) a matrix (M) protein of the coronavirus, (c) anucleocapsid (N) protein of the coronavirus, (d) an envelope (E) proteinof the coronavirus, (e) a hemagglutinin-esterase (HE) protein, (f) animmunogenic fragment of any one of (a) to (e); and (g) a functionalderivative of any one of (a) to (f).

In some embodiments, the viral peptide or protein is the S protein, animmunogenic fragment of the S protein, or a functional derivative of theS protein or the immunogenic fragment thereof. In some embodiments, theimmunogenic fragment of the S protein is selected from an ectodomain(ECD), an S1 subunit, a receptor binding domain (RBD), and areceptor-binding motif (RBM).

In some embodiments of the pharmaceutical composition described herein,the viral peptide or protein is a functional derivative of RBD. In someembodiments, the functional derivative of RBD comprises one or moreamino acid substitutions in the RBD that are capable of increasingbinding affinity of the RBD to receptor in a host cell. In someembodiments, the receptor is ACE2. In some embodiments, the amino acidsubstitution comprises N501T.

In some embodiments of the pharmaceutical composition described herein,the viral peptide or protein comprises the amino acid sequence set forthin SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16 18, 20-26, 32, 34, 40, 42, and44. In some embodiments, the nucleic acid comprises the sequence as setforth in SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 46, 48, 50, 52, 54, 56 or a transcribed RNA sequencethereof.

In some embodiments of the pharmaceutical composition described herein,the functional derivative of the RBD comprises the RBD fused to an Fcregion of human immunoglobulin. In some embodiments, the immunoglobulinis IgG1.

In some embodiments of the pharmaceutical composition described herein,the functional derivative of RBD comprises the RBD fused to a peptidefacilitating multimerization of the fusion protein. In some embodiments,the functional derivative of S-RBD is configured to form a trimericcomplex.

In some embodiments of the pharmaceutical composition described herein,the viral peptide or protein is the N protein. In some embodiments, theN protein comprises the amino acid sequence set forth in SEQ ID NO: 18.In some embodiments, the nucleic acid comprises the sequence set forthin SEQ ID NO: 19 or a RNA sequence transcribed therefrom.

In some embodiments of the pharmaceutical composition described herein,the nucleic acid further comprises a 5′ untranslated region and/or a 3′untranslated region. In some embodiments, the 5′ untranslated regioncomprises a sequence selected from SEQ ID NOS: 46 51. In someembodiments, the 3′ untranslated region comprises a poly-A tail or apolyadenylation signal. In some embodiments, the 3′ untranslated regioncomprises a sequence selected from SEQ ID NOS: 52-57.

In some embodiments of the pharmaceutical composition described herein,the nucleic acid comprises one or more functional nucleotide analogsselected from pseudouridine, 1-methyl-pseudouridine and5-methylcytosine.

In some embodiments of the pharmaceutical composition described herein,the composition further comprises at least one lipid. In someembodiments, the lipid is a compound according to Formula (I) to (IV).In some embodiments, the lipid is a compound according to Formula (I-A),(I-B), (IB′), (I-B″), (I-C), (I-D), (I-E), (I-F), (I-F′), (I-F″), (I-G),(I-H), (I-I), (I-J), (I-J′), (I-J″), (I-K), (I-L), (I-M), (I-N), (I-N′),(I-N″), (I-O), (I-P), (I-Q), (I-R), (I-R′), (I-R″), (I S), (I-T), (I-U),(II-A), (II-B), (II-B′), (II-B″), (II-C), (II-D), (II-E), (II-F),(II-F′), (II-F″), (II-G), (II-H), (II-I), (II-J), (II-J′), (II-J″),(II-K), (II-L), (II-M), (II-N), (II-N′), (II-N″), (II-O), (II-P),(II-Q), (II-R), (II-R′), (II-R″), (II-S), (II-T), (II-U), (III-A),(III-B), (III-B′), (III-B″), (III-C), (III-D), (III-E), (III-F),(III-F′), (III-F″), (III-G), (III-H), (III-I), (III-J), (III-J′),(III-J″), (III-K), (III-L), (III-M), (III-N), (III-N′), (III-N″),(III-O), (III-P), (III-Q), (III-R), (III-R′), (III-R″), (III-S),(III-T), (III-U), (IV-A), (IV-B), (IV-B′), (IV-B″), (IV-C), (IV-D),(IV-E), (IV-F), (IV-F′), (IV-F″), (IV-G), (IV-H), (IV-I), (IV-J),(IV-J′), (IV-J″), (IV-K), (IV-L), (IV-M), (IV-N), (IV-N′), (IV-N″),(IV-O), (IV-P), (IV-Q), (IV-R), (IV-R′), (IV-R″), (IV-S), (IV-T) or(IV-U). In some embodiments, the lipid is a compound listed in Table 1.In some embodiments, the composition is formulated as lipidnanoparticles encapsulating the nucleic acid in a lipid shell. In someembodiments, the composition is a vaccine.

In one aspect, provided herein are methods for managing, preventing ortreating an infectious disease caused by coronavirus in a subject,comprising administering to the subject a therapeutically effectiveamount of the non-naturally occurring nucleic acid described herein, ora therapeutically effective amount of the pharmaceutical composition asdescribed herein.

In some embodiments of the method described herein, the subject is ahuman or a non-human mammal. In some embodiments, the subject is a humanadult, a human child or a human toddler. In some embodiments, thesubject has the infectious disease. In some embodiments, the subject isat risk of, or is susceptible to, infection by the coronavirus. In someembodiments, the subject is an elderly human. In some embodiments,subject has been diagnosed positive for infection by the coronavirus. Insome embodiments, the subject is asymptomatic.

In some embodiments of the method described herein, the method comprisesadministering lipid nanoparticles encapsulating the nucleic acid to thesubject, and wherein the lipid nanoparticles are endocytosed by thecells in the subject. In some embodiments, the nucleic acid is expressedby the cells in the subject.

In some embodiments of the method described herein, an immune responseagainst the coronavirus is elicited in the subject. In some embodiments,the immune response comprises production of an antibody specificallybinds to the viral peptide or protein encoded by the nucleic acid. Insome embodiments, the antibody is a neutralizing antibody against thecoronavirus or cells infected by the coronavirus. In some embodiments,the serum titer of the antibody is increased in the subject.

In some embodiments, the antibody specifically binds to one or moreepitopes of the S protein. In some embodiments of the method describedherein, one or more function or activity of the S protein is attenuated.In some embodiments, the attenuation of the S protein function oractivity is measured by (a) reduction of binding of the S protein tohost cell receptor; (b) reduction of attachment of the coronavirus to ahost cell; (c) reduction of host cell membrane fusion induced by thecoronavirus; or (d) reduction of the number of cells infected by thecoronavirus in the subject. In some embodiments, the host receptor isselected from angiotensin-converting enzyme 2 (ACE2), aminopeptidase N(APN), dipeptidyl peptidase 4 (DPP4), carcinoembryonic antigen-relatedcell adhesion molecule 1 (CEACAM1) and sugar. In some embodiments, the Sprotein function or activity is reduced by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or 100%.

In some embodiments of the method described herein, the antibodyspecifically bind to one or more epitopes of the N protein. In someembodiments, one or more function or activity of the N protein isattenuated. In some embodiments, the attenuation of the N proteinfunction or activity is measured by (a) reduction of binding of the Nprotein to replicated genomic sequence of the coronavirus; (b) reductionof packaging of replicated genomic sequence of the coronavirus into afunctional viral capsid; or (c) reduction of the number of replicatedviral particles in the subject. In some embodiments, the N proteinfunction or activity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 100%.

In some embodiments of the method described herein, antibody binds to aviral particle or an infected cell and mark the viral particle ofinfected cell for destruction by the immune system of the subject. Insome embodiments, endocytosis of viral particles bound by the antibodyis induced or enhanced. In some embodiments, antibody-dependentcell-mediated cytotoxicity (ADCC) against infected cells in the subjectis induced or enhanced. In some embodiments, antibody-dependent cellularphagocytosis (ADCP) against infected cells in the subject is induced orenhanced. In some embodiments, complement dependent cytotoxicity (CDC)against infected cells in the subject is induced or enhanced.

In some embodiments of the method described herein, the infectiousdisease is respiratory tract infection, lung infection, renal infection,liver infection, enteric infection, neurologic infections, respiratorysyndrome, bronchitis, pneumonia, gastroenteritis, encephalomyelitis,encephalitis, sarcoidosis, diarrhea, hepatitis, and demyelinatingdisease. In some embodiments, the infectious disease is respiratorytract infection. In some embodiments, the infectious disease is lunginfection. In some embodiments, the infectious disease is respiratorysyndrome. In some embodiments, the infectious disease is pneumonia.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an exemplary HPLC analysis and purification of in vitrotranscribed mRNA constructs according to the present disclosure. Themain peak (b) represents in vitro transcribed mRNA molecules, and theminor peak (a) represents an entity of impurity.

FIG. 2 shows confocal fluorescence microscopy images of Hela cellstransfected with mRNA constructs according to the present disclosure.The RBD-FITC channel shows staining of the cells using 3 differentmonoclonal antibodies (H014, mh001 and mh219) recognizing SARS-CoV-2 Sprotein RBD, respectively. The DAPI channel shows staining of the cellswith the blue-fluorescent DNA stain DAPI(4′,6-diamidino-2-phenylindole). The Bright channel shows bright fieldimages of the cells. Non-transfected Hela cells (Mock) was included as anegative control. Scale bar is 50 mm.

FIG. 3 shows Western blot analysis of culture supernatant of HeLa cellstransfected with mRNA constructs encoding SARS-CoV-2 S protein antigensaccording to the present disclosure. Particularly, three different mRNAconstructs encoding different antigenic fragments of the SARS-CoV-2 Sprotein RBD (RBD sample-1, RBD sample-2 and rRBD-His) were included inthe analysis. An irrelevant mRNA control was also included. Monomers anddimmers of the encoded RBD fragments are shown on the blot.

FIG. 4 shows exemplary quantification of mRNA-encoded SARS-CoV-2 Sprotein antigen concentrations (ng/mL) of in cell culture supernatant asdetermined by ELISA.

FIG. 5 shows neutralizing antibody titers in sera collected from micevaccinated with lipid nanoparticles (LNP) containing mRNA encoding aSARS-CoV-2 antigen. Particularly, neutralizing antibody titer wasmeasured as the PRNT50 value.

FIG. 6 shows the RBD expression levels in the serums of five groups ofexperimental mice which received 1 ug-5 ug dosing amounts.

FIG. 7 shows the detection results of RBD-specific IgG antibody titersin immunized mice on days 14, 21 and 29 as measured by ELISA.

6. DETAILED DESCRIPTION

Provided herein are therapeutic nucleic acid molecules useful for theprevention, management and treatment of infectious disease or conditioncaused by coronaviruses. Also provided herein are pharmaceuticalcomposition comprising the therapeutic nucleic acid molecules, includingpharmaceutical composition formulated as lipid nanoparticles and relatedtherapeutic methods and uses for preventing, managing and treating ofinfectious disease or condition caused by coronaviruses, including thepathogen causing the pandemic known as the COVID-19. Additional featuresof the present disclosure will become apparent to those skilled in theart upon consideration of the following detailed description ofparticular embodiments.

6.1 General Techniques

Techniques and procedures described or referenced herein include thosethat are generally well understood and/or commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized methodologies described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocolsin Molecular Biology (Ausubel et al. eds., 2003).

6.2 Terminology

Unless described otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one ofordinary skill in the art. For purposes of interpreting thisspecification, the following description of terms will apply andwhenever appropriate, terms used in the singular will also include theplural and vice versa. All patents, applications, publishedapplications, and other publications are incorporated by reference intheir entirety. In the event that any description of terms set forthconflicts with any document incorporated herein by reference, thedescription of term set forth below shall control.

As used herein and unless otherwise specified, the term “lipid” refersto a group of organic compounds that include, but are not limited to,esters of fatty acids and are generally characterized by being poorlysoluble in water, but soluble in many nonpolar organic solvents. Whilelipids generally have poor solubility in water, there are certaincategories of lipids (e.g., lipids modified by polar groups, e.g.,DMG-PEG2000) that have limited aqueous solubility and can dissolve inwater under certain conditions. Known types of lipids include biologicalmolecules such as fatty acids, waxes, sterols, fat-soluble vitamins,monoglycerides, diglycerides, triglycerides, and phospholipids. Lipidscan be divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3)“derived lipids” such as steroids. Further, as used herein, lipids alsoencompass lipidoid compounds. The term “lipidoid compound,” also simply“lipidoid”, refers to a lipid-like compound (e.g. an amphiphiliccompound with lipid-like physical properties).

The term “lipid nanoparticle” or “LNP” refers to a particle having atleast one dimension on the order of nanometers (nm) (e.g., 1 to 1,000nm), which contains one or more types of lipid molecules. The LNPprovided herein can further contain at least one non-lipid payloadmolecule (e.g., one or more nucleic acid molecules). In someembodiments, the LNP comprises a non-lipid payload molecule eitherpartially or completely encapsulated inside a lipid shell. Particularly,in some embodiments, wherein the payload is a negatively chargedmolecule (e.g., mRNA encoding a viral protein), and the lipid componentsof the LNP comprise at least one cationic lipid. Without being bound bythe theory, it is contemplated that the cationic lipids can interactwith the negatively charged payload molecules and facilitatesincorporation and/or encapsulation of the payload into the LNP duringLNP formation. Other lipids that can form part of a LNP as providedherein include but are not limited to neutral lipids and charged lipids,such as steroids, polymer conjugated lipids, and various zwitterioniclipids. In certain embodiments, a LNP according to the presentdisclosure comprises one or more lipids of Formula (I) to (IV) (andsub-formulas thereof) as described herein.

The term “cationic lipid” refers to a lipid that is either positivelycharged at any pH value or hydrogen ion activity of its environment, orcapable of being positively charged in response to the pH value orhydrogen ion activity of its environment (e.g., the environment of itsintended use). Thus, the term “cationic” encompasses both “permanentlycationic” and “cationisable.” In certain embodiments, the positivecharge in a cationic lipid results from the presence of a quaternarynitrogen atom. In certain embodiments, the cationic lipid comprises azwitterionic lipid that assumes a positive charge in the environment ofits intended use (e.g., at physiological pH). In certain embodiments,the cationic lipid is one or more lipids of Formula (I) to (IV) (andsub-formulas thereof) as described herein.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid (PEG-lipid), in which the polymerportion comprises a polyethylene glycol.

The term “neutral lipid” encompasses any lipid molecules existing inuncharged forms or neutral zwitterionic forms at a selected pH value orwithin a selected pH range. In some embodiments, the selected useful pHvalue or range corresponds to the pH condition in an environment of theintended uses of the lipids, such as the physiological pH. Asnon-limiting examples, neutral lipids that can be used in connectionwith the present disclosure include, but are not limited to,phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate(DOCP), sphingomyelins (SM), ceramides, steroids such as sterols andtheir derivatives. Neutral lipids as provided herein may be synthetic orderived (isolated or modified) from a natural source or compound.

The term “charged lipid” encompasses any lipid molecules that exist ineither positively charged or negatively charged forms at a selected pHor within a selected pH range. In some embodiments, the selected pHvalue or range corresponds to the pH condition in an environment of theintended uses of the lipids, such as the physiological pH. Asnon-limiting examples, neutral lipids that can be used in connectionwith the present disclosure include, but are not limited to,phosphatidylserines, phosphatidic acids, phosphatidylglycerols,phosphatidylinositols, sterol hemisuccinates, dialkyltrimethylarnmonium-propanes, (e.g., DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g., DC-Chol),1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt(DOPG-Na), and 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt(DOPA-Na). Charged lipids as provided herein may be synthetic or derived(isolated or modified) from a natural source or compound.

As used herein, and unless otherwise specified, the term “alkyl” refersto a straight or branched hydrocarbon chain radical consisting solely ofcarbon and hydrogen atoms, which is saturated. In one embodiment, thealkyl group has, for example, from one to twenty-four carbon atoms(C₁-C₂₄ alkyl), four to twenty carbon atoms (C₄-C₂₀ alkyl), six tosixteen carbon atoms (C₆-C₁₆ alkyl), six to nine carbon atoms (C₆-C₉alkyl), one to fifteen carbon atoms (C₁-C₁₅ alkyl), one to twelve carbonatoms (C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₈ alkyl) or one tosix carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of themolecule by a single bond. Examples of alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl,n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, andthe like. Unless otherwise specified, an alkyl group is optionallysubstituted.

As used herein, and unless otherwise specified, the term “alkenyl”refers to a straight or branched hydrocarbon chain radical consistingsolely of carbon and hydrogen atoms, which contains one or morecarbon-carbon double bonds. The term “alkenyl” also embraces radicalshaving “cis” and “trans” configurations, or alternatively, “E” and “Z”configurations, as appreciated by those of ordinary skill in the art. Inone embodiment, the alkenyl group has″ for example, from two totwenty-four carbon atoms (C₂-C₂₄ alkenyl), four to twenty carbon atoms(C₄-C₂₀ alkenyl), six to sixteen carbon atoms (C₆-C₁₆ alkenyl), six tonine carbon atoms (C₆-C₉ alkenyl), two to fifteen carbon atoms (C₂-C₁₅alkenyl), two to twelve carbon atoms (C₂-C₁₂ alkenyl), two to eightcarbon atoms (C₂-C₈ alkenyl) or two to six carbon atoms (C₂-C₆ alkenyl)and which is attached to the rest of the molecule by a single bond.Examples of alkenyl groups include, but are not limited to, ethenyl,prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.Unless otherwise specified, an alkenyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkynyl”refers to a straight or branched hydrocarbon chain radical consistingsolely of carbon and hydrogen atoms, which contains one or morecarbon-carbon triple bonds. In one embodiment, the alkynyl group has,for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkynyl), fourto twenty carbon atoms (C₄-C₂₀ alkynyl), six to sixteen carbon atoms(C₆-C₁₆ alkynyl), six to nine carbon atoms (C₆-C₉ alkynyl), two tofifteen carbon atoms (C₂-C₁₅ alkynyl), two to twelve carbon atoms(C₂-C₁₂ alkynyl), two to eight carbon atoms (C₂-C₈ alkynyl) or two tosix carbon atoms (C₂-C₆ alkynyl) and which is attached to the rest ofthe molecule by a single bond. Examples of alkynyl groups include, butare not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like.Unless otherwise specified, an alkynyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkylene” or“alkylene chain” refers to a straight or branched divalent hydrocarbonchain linking the rest of the molecule to a radical group, consistingsolely of carbon and hydrogen, which is saturated. In one embodiment,the alkylene has, for example, from one to twenty-four carbon atoms(C₁-C₂₄ alkylene), one to fifteen carbon atoms (C₁-C₁₅ alkylene),one totwelve carbon atoms (C₁-C₁₂ alkylene), one to eight carbon atoms (C₁-C₈alkylene), one to six carbon atoms (C₁-C₆ alkylene), two to four carbonatoms (C₂-C₄ alkylene), one to two carbon atoms (C₁-C₂ alkylene).Examples of alkylene groups include, but are not limited to, methylene,ethylene, propylene, n-butylene, and the like. The alkylene chain isattached to the rest of the molecule through a single bond and to theradical group through a single bond. The points of attachment of thealkylene chain to the rest of the molecule and to the radical group canbe through one carbon or any two carbons within the chain. Unlessotherwise specified, an alkylene chain is optionally substituted.

As used herein, and unless otherwise specified, the term “alkenylene”refers to a straight or branched divalent hydrocarbon chain linking therest of the molecule to a radical group, consisting solely of carbon andhydrogen, which contains one or more carbon-carbon double bonds. In oneembodiment, the alkenylene has, for example, from two to twenty-fourcarbon atoms (C₂-C₂₄ alkenylene), two to fifteen carbon atoms (C₂-C₁₅alkenylene), two to twelve carbon atoms (C₂-C₁₂ alkenylene), two toeight carbon atoms (C₂-C₈ alkenylene), two to six carbon atoms (C₂-C₆alkenylene) or two to four carbon atoms (C₂-C₄ alkenylene). Examples ofalkenylene include, but are not limited to, ethenylene, propenylene,n-butenylene, and the like. The alkenylene is attached to the rest ofthe molecule through a single or double bond and to the radical groupthrough a single or double bond. The points of attachment of thealkenylene to the rest of the molecule and to the radical group can bethrough one carbon or any two carbons within the chain. Unless otherwisespecified, an alkenylene is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkyl”refers to a non-aromatic monocyclic or polycyclic hydrocarbon radicalconsisting solely of carbon and hydrogen atoms, and which is saturated.Cycloalkyl group may include fused or bridged ring systems. In oneembodiment, the cycloalkyl has, for example, from 3 to 15 ring carbonatoms (C₃-C₁₅ cycloalkyl), from 3 to 10 ring carbon atoms (C₃-C₁₀cycloalkyl), or from 3 to 8 ring carbon atoms (C₃-C₈ cycloalkyl). Thecycloalkyl is attached to the rest of the molecule by a single bond.Examples of monocyclic cycloalkyl radicals include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. Examples of polycyclic cycloalkyl radicals include, but arenot limited to, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwisespecified, a cycloalkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkylene”is a divalent cycloalkyl group. Unless otherwise specified, acycloalkylene group isoptionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkenyl”refers to a non-aromatic monocyclic or polycyclic hydrocarbon radicalconsisting solely of carbon and hydrogen atoms, and which includes oneor more carbon-carbon double bonds. Cycloalkenyl may include fused orbridged ring systems. In one embodiment, the cycloalkenyl has, forexample, from 3 to 15 ring carbon atoms (C₃-C₁₅ cycloalkenyl), from 3 to10 ring carbon atoms (C₃-C₁₀ cycloalkenyl), or from 3 to 8 ring carbonatoms (C₃-C₈ cycloalkenyl). The cycloalkenyl is attached to the rest ofthe molecule by a single bond. Examples of monocyclic cycloalkenylradicals include, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.Unless otherwise specified, a cycloalkenyl group is optionallysubstituted.

As used herein, and unless otherwise specified, the term“cycloalkenylene” is a divalent cycloalkenyl group. Unless otherwisespecified, a cycloalkenylene group is optionally substituted.

As used herein, and unless otherwise specified, the term “heterocyclyl”refers to a non-aromatic radical monocyclic or polycyclic moiety thatcontains one or more (e.g., one, one or two, one to three, or one tofour) heteroatoms independently selected from nitrogen, oxygen,phosphorous, and sulfur. The heterocyclyl may be attached to the mainstructure at any heteroatom or carbon atom. A heterocyclyl group can bea monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic ringsystem, wherein the polycyclic ring systems can be a fused, bridged orspiro ring system. Heterocyclyl polycyclic ring systems can include oneor more heteroatoms in one or more rings. A heterocyclyl group can besaturated or partially unsaturated. Saturated heterocycloalkyl groupscan be termed “heterocycloalkyl”. Partially unsaturated heterocycloalkylgroups can be termed “heterocycloalkenyl” if the heterocyclyl containsat least one double bond, or “heterocycloalkynyl” if the heterocyclylcontains at least one triple bond. In one embodiment, the heterocyclylhas, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms(3- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-memberedheterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl).Whenever it appears herein, a numerical range such as “3 to 18” refersto each integer in the given range; e.g., “3 to 18 ring atoms” meansthat the heterocyclyl group can consist of 3 ring atoms, 4 ring atoms, 5ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10ring atoms, etc., up to and including 18 ring atoms. Examples ofheterocyclyl groups include, but are not limited to, imidazolyl,imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl,pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl,isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl,tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, andisoquinolyl. Unless otherwise specified, a heterocyclyl group isoptionally substituted.

As used herein, and unless otherwise specified, the term“heterocyclylene” is a divalent heterocyclyl group. Unless otherwisespecified, a heterocyclylene group is optionally substituted

As used herein, and unless otherwise specified, the term “aryl” refersto a monocyclic aromatic group and/or multicyclic monovalent aromaticgroup that contain at least one aromatic hydrocarbon ring. In certainembodiments, the aryl has from 6 to 18 ring carbon atoms (C₆-C₁₈ aryl),from 6 to 14 ring carbon atoms (C₆-C₁₄ aryl), or from 6 to 10 ringcarbon atoms (C₆-C₁₀ aryl). Examples of aryl groups include, but are notlimited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl,pyrenyl, biphenyl, and terphenyl. The term “aryl” also refers tobicyclic, tricyclic, or other multicyclic hydrocarbon rings, where atleast one of the rings is aromatic and the others of which may besaturated, partially unsaturated, or aromatic, for example,dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl).Unless otherwise specified, an aryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “arylene” is adivalent aryl group. Unless otherwise specified, an arylene group isoptionally substituted.

As used herein, and unless otherwise specified, the term “heteroaryl”refers to a monocyclic aromatic group and/or multicyclic aromatic groupthat contains at least one aromatic ring, wherein at least one aromaticring contains one or more (e.g., one, one or two, one to three, or oneto four) heteroatoms independently selected from O, S, and N. Theheteroaryl may be attached to the main structure at any heteroatom orcarbon atom. In certain embodiments, the heteroaryl has from 5 to 20,from 5 to 15, or from 5 to 10 ring atoms. The term “heteroaryl” alsorefers to bicyclic, tricyclic, or other multicyclic rings, where atleast one of the rings is aromatic and the others of which may besaturated, partially unsaturated, or aromatic, wherein at least onearomatic ring contains one or more heteroatoms independently selectedfrom O, S, and N. Examples of monocyclic heteroaryl groups include, butare not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl,thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, andtriazinyl. Examples of bicyclic heteroaryl groups include, but are notlimited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl,quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl,benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl,coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl,pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, andtetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include,but are not limited to, carbazolyl, benzindolyl, phenanthrollinyl,acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise specified, aheteroaryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “heteroarylene”is a divalent heteroaryl group. Unless otherwise specified, aheteroarylene group is optionally substituted.

When the groups described herein are said to be “substituted,” they maybe substituted with any appropriate substituent or substituents.Illustrative examples of substituents include, but are not limited to,those found in the exemplary compounds and embodiments provided herein,as well as: a halogen atom such as F, Cl, Br, or I; cyano; oxo (═O);hydroxyl (OH); alkyl; alkenyl; alkynyl; cycloalkyl; aryl; —(C═O)OR′;—O(C═O)R′; —C(═O)R′; —OR′; —S(O)_(x)R′; —S—SR′; —C(═O)SR′; —SC(═O)R′;—NR′R′; —NR′C(═O)R′; —C(═O)NR′R′; —NR′ C(═O)NR′R′; —OC(═O)NR′R′;—NR′C(═O)OR′; —NR′ S(O)_(x)NR′R′; —NR S(O)_(x)R′; and —S(O)_(x)NR′R′,wherein: R′ is, at each occurrence, independently H, C₁-C₁₅ alkyl orcycloalkyl, and x is 0, 1 or 2. In some embodiments the substituent is aC₁-C₁₂ alkyl group. In other embodiments, the substituent is acycloalkyl group. In other embodiments, the substituent is a halo group,such as fluoro. In other embodiments, the substituent is an oxo group.In other embodiments, the substituent is a hydroxyl group. In otherembodiments, the substituent is an alkoxy group (—OR′). In otherembodiments, the substituent is a carboxyl group. In other embodiments,the substituent is an amino group (—NR′R′).

As used herein, and unless otherwise specified, the term “optional” or“optionally” (e.g., optionally substituted) means that the subsequentlydescribed event of circumstances may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means that the alkyl radical may or may not be substituted andthat the description includes both substituted alkyl radicals and alkylradicals having no substitution.

As used herein, and unless otherwise specified, the term “prodrug” of abiologically active compound refers to a compound that may be convertedunder physiological conditions or by solvolysis to the biologicallyactive compound. In one embodiment, the term “prodrug” refers to ametabolic precursor of the biologically active compound that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to thebiologically active compound. Prodrugs are typically rapidly transformedin vivo to yield the parent biologically active compound, for example,by hydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7 9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

In one embodiment, the term “prodrug” is also meant to include anycovalently bonded carriers, which release the active compound in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound may be prepared by modifying functional groups present in thecompound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds wherein a hydroxyl, amino or mercapto group is bondedto any group that, when the prodrug of the compound is administered to amammalian subject, cleaves to form a free hydroxyl, free amino or freemercapto group, respectively.

Examples of prodrugs include, but are not limited to, acetate, formateand benzoate derivatives of alcohol or amide derivatives of aminefunctional groups in the compounds provided herein.

As used herein, and unless otherwise specified, the term“pharmaceutically acceptable salt” includes both acid and base additionsalts.

Examples of pharmaceutically acceptable acid addition salts include, butare not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid and the like, and organic acids such as,but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid,alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid,benzoic acid, 4-acetamidobenzoic acid, camphoric acid,camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid,carbonic acid, cinnamic acid, citric acid, cyclamic acid,dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaricacid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid,glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoricacid, glycolic acid, hippuric acid, isobutyric acid, lactic acid,lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid,mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

Examples of pharmaceutically acceptable base addition salt include, butare not limited to, salts prepared from addition of an inorganic base oran organic base to a free acid compound. Salts derived from inorganicbases include, but are not limited to, the sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. In one embodiment, the inorganic salts are theammonium, sodium, potassium, calcium, and magnesium salts. Salts derivedfrom organic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as ammonia, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. In one embodiment, the organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

A compound provided herein may contain one or more asymmetric centersand may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids.Unless otherwise specified, a compound provided herein is meant toinclude all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

As used herein, and unless otherwise specified, the term “isomer” refersto different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space. “Atropisomers” are stereoisomers from hinderedrotation about single bonds. “Enantiomers” are a pair of stereoisomersthat are non-superimposable mirror images of each other. A mixture of apair of enantiomers in any proportion can be known as a “racemic”mixture. “Diastereoisomers” are stereoisomers that have at least twoasymmetric atoms, but which are not mirror-images of each other.

“Stereoisomers” can also include E and Z isomers, or a mixture thereof,and cis and trans isomers or a mixture thereof. In certain embodiments,a compound described herein is isolated as either the E or Z isomer. Inother embodiments, a compound described herein is a mixture of the E andZ isomers.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The concentrations of the isomeric formswill depend on the environment the compound is found in and may bedifferent depending upon, for example, whether the compound is a solidor is in an organic or aqueous solution.

It should also be noted a compound described herein can containunnatural proportions of atomic isotopes at one or more of the atoms.For example, the compounds may be radiolabeled with radioactiveisotopes, such as for example tritium (³H), iodine-125 (¹²⁵I), sulfur-35(³⁵S), or carbon-14 (¹⁴C), or may be isotopically enriched, such as withdeuterium (²H), carbon-13 (¹³C), or nitrogen-15 (¹⁵N). As used herein,an “isotopolog” is an isotopically enriched compound. The term“isotopically enriched” refers to an atom having an isotopic compositionother than the natural isotopic composition of that atom. “Isotopicallyenriched” may also refer to a compound containing at least one atomhaving an isotopic composition other than the natural isotopiccomposition of that atom. The term “isotopic composition” refers to theamount of each isotope present for a given atom. Radiolabeled andisotopically enriched compounds are useful as therapeutic agents, e.g.,cancer therapeutic agents, research reagents, e.g., binding assayreagents, and diagnostic agents, e.g., in vivo imaging agents. Allisotopic variations of a compound described herein, whether radioactiveor not, are intended to be encompassed within the scope of theembodiments provided herein. In some embodiments, there are providedisotopologs of a compound described herein, for example, the isotopologsare deuterium, carbon-13, and/or nitrogen-15 enriched. As used herein,“deuterated”, means a compound wherein at least one hydrogen (H) hasbeen replaced by deuterium (indicated by D or ²H), that is, the compoundis enriched in deuterium in at least one position.

It should be noted that if there is a discrepancy between a depictedstructure and a name for that structure, the depicted structure is to beaccorded more weight.

As used herein, and unless otherwise specified, the term“pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

The term “composition” is intended to encompass a product containing thespecified ingredients (e.g., a mRNA molecule provided herein) in,optionally, the specified amounts.

The term “polynucleotide” or “nucleic acid,” as used interchangeablyherein, refers to polymers of nucleotides of any length and includes,e.g., DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase or by a synthetic reaction. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and their analogs.Nucleic acid can be in either single- or double-stranded forms. As usedherein and unless otherwise specified, “nucleic acid” also includesnucleic acid mimics such as locked nucleic acids (LNAs), peptide nucleicacids (PNAs), and morpholinos. “Oligonucleotide,” as used herein, refersto short synthetic polynucleotides that are generally, but notnecessarily, fewer than about 200 nucleotides in length. The terms“oligonucleotide” and “polynucleotide” are not mutually exclusive. Thedescription above for polynucleotides is equally and fully applicable tooligonucleotides. Unless specified otherwise, the left-hand end of anysingle-stranded polynucleotide sequence disclosed herein is the 5′ end;the left-hand direction of double-stranded polynucleotide sequences isreferred to as the 5′ direction. The direction of 5′ to 3′ addition ofnascent RNA transcripts is referred to as the transcription direction;sequence regions on the DNA strand having the same sequence as the RNAtranscript that are 5′ to the 5′ end of the RNA transcript are referredto as “upstream sequences”; sequence regions on the DNA strand havingthe same sequence as the RNA transcript that are 3′ to the 3′ end of theRNA transcript are referred to as “downstream sequences.”

As used herein, the term “non-naturally occurring” when used inreference to a nucleic acid molecule as described herein is intended tomean that the nucleic acid molecule is not found in nature. Anon-naturally occurring nucleic acid encoding a viral peptide or proteincontains at least one genetic alternation or chemical modification notnormally found in a naturally occurring strain of the virus, includingwild-type strains of the virus. Genetic alterations include, forexample, modifications introducing expressible nucleic acid sequencesencoding peptides or polypeptides heterologous to the virus, othernucleic acid additions, nucleic acid deletions, nucleic acidsubstitution, and/or other functional disruption of the virus' geneticmaterial. Such modifications include, for example, modifications in thecoding regions and functional fragments thereof, for heterologous,homologous or both heterologous and homologous polypeptides for theviral species. Additional modifications include, for example,modifications in non-coding regulatory regions in which themodifications alter expression of a gene or operon. Additionalmodifications also include, for example, incorporation of a nucleic acidsequence into a vector, such as a plasmid or an artificial chromosome.Chemical modifications include, for example, one or more functionalnucleotide analog as described herein.

An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA,or a mixed nucleic acids, which is substantially separated from othergenome DNA sequences as well as proteins or complexes such as ribosomesand polymerases, which naturally accompany a native sequence. An“isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as an mRNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. In a specific embodiment, one or more nucleicacid molecules encoding an antigen as described herein are isolated orpurified. The term embraces nucleic acid sequences that have beenremoved from their naturally occurring environment, and includesrecombinant or cloned DNA or RNA isolates and chemically synthesizedanalogues or analogues biologically synthesized by heterologous systems.A substantially pure molecule may include isolated forms of themolecule.

The term “encoding nucleic acid” or grammatical equivalents thereof asit is used in reference to nucleic acid molecule encompasses (a) anucleic acid molecule in its native state or when manipulated by methodswell known to those skilled in the art that can be transcribed toproduce mRNA which is then translated into a peptide and/or polypeptide,and (b) the mRNA molecule itself. The antisense strand is the complementof such a nucleic acid molecule, and the encoding sequence can bededuced therefrom. The term “coding region” refers to a portion in anencoding nucleic acid sequence that is translated into a peptide orpolypeptide. The term “untranslated region” or “UTR” refers to theportion of an encoding nucleic acid that is not translated into apeptide or polypeptide. Depending on the orientation of a UTR withrespect to the coding region of a nucleic acid molecule, a UTR isreferred to as the 5′-UTR if located to the 5′-end of a coding region,and a UTR is referred to as the 3′-UTR if located to the 3′-end of acoding region.

The term “mRNA” as used herein refers to a message RNA moleculecomprising one or more open reading frame (ORF) that can be translatedby a cell or an organism provided with the mRNA to produce one or morepeptide or protein product. The region containing the one or more ORFsis referred to as the coding region of the mRNA molecule. In certainembodiments, the mRNA molecule further comprises one or moreuntranslated regions (UTRs).

In certain embodiments, the mRNA is a monocistronic mRNA that comprisesonly one ORF. In certain embodiments, the monocistronic mRNA encodes apeptide or protein comprising at least one epitope of a selected antigen(e.g., a pathogenic antigen or a tumor associated antigen). In otherembodiments, the mRNA is a multicistronic mRNA that comprises two ormore ORFs. In certain embodiments, the multiecistronic mRNA encodes twoor more peptides or proteins that can be the same or different from eachother. In certain embodiments, each peptide or protein encoded by amulticistronic mRNA comprises at least one epitope of a selectedantigen. In certain embodiments, different peptide or protein encoded bya multicistronic mRNA each comprises at least one epitope of differentantigens. In any of the embodiments described herein, the at least oneepitope can be at least 2, at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, or at least 10 epitopes of anantigen.

The term “nucleobases” encompasses purines and pyrimidines, includingnatural compounds adenine, thymine, guanine, cytosine, uracil, inosine,and natural or synthetic analogs or derivatives thereof.

The term “functional nucleotide analog” as used herein refers to amodified version of a canonical nucleotide A, G, C, U or T that (a)retains the base-pairing properties of the corresponding canonicalnucleotide, and (b) contains at least one chemical modification to (i)the nucleobase, (ii) the sugar group, (iii) the phosphate group, or (iv)any combinations of (i) to (iii), of the corresponding naturalnucleotide. As used herein, base pairing encompasses not only thecanonical Watson-Crick adenine-thymine, adenine-uracil, orguanine-cytosine base pairs, but also base pairs formed betweencanonical nucleotides and functional nucleotide analogs or between apair of functional nucleotide analogs, wherein the arrangement ofhydrogen bond donors and hydrogen bond acceptors permits hydrogenbonding between a modified nucleobase and a canonical nucleobase orbetween two complementary modified nucleobase structures. For example, afunctional analog of guanosine (G) retains the ability to base-pair withcytosine (C) or a functional analog of cytosine. One example of suchnon-canonical base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine, or uracil. As describedherein, a functional nucleotide analog can be either naturally occurringor non-naturally occurring. Accordingly, a nucleic acid moleculecontaining a functional nucleotide analog can have at least one modifiednucleobase, sugar group and/or internucleoside linkage. Exemplarychemical modifications to the nucleobases, sugar groups, orinternucleoside linkages of a nucleic acid molecule are provided herein.

The terms “translational enhancer element,” “TEE” and “translationalenhancers” as used herein refers to an region in a nucleic acid moleculethat functions to promotes translation of a coding sequence of thenucleic acid into a protein or peptide product, such as viacap-dependent or cap-independent translation. A TEE typically locates inthe UTR region of a nucleic acid molecule (e.g., mRNA) and enhance thetranslational level of a coding sequence located either upstream ordownstream. For example, a TEE in a 5′-UTR of a nucleic acid moleculecan locate between the promoter and the starting codon of the nucleicacid molecule. Various TEE sequences are known in the art (Wellensiek etal. Genome-wide profiling of human cap-independent translation-enhancingelements, Nature Methods, 2013 August; 10(8): 747-750; Chappell et al.PNAS Jun. 29, 2004 101 (26) 9590-9594). Some TEEs are known to beconserved across multiple species (Panek et al. Nucleic Acids Research,Volume 41, Issue 16, 1 Sep. 2013, Pages 7625-7634).

As used herein, the term “stem-loop sequence” refers to asingle-stranded polynucleotide sequence having at least two regions thatare complementary or substantially complementary to each other when readin opposite directions, and thus capable of base-pairing with each otherto form at least one double helix and an unpaired loop. The resultingstructure is known as a stem-loop structure, a hairpin, or a hairpinloop, which is a secondary structure found in many RNA molecules.

The term “peptide” as used herein refers to a polymer containing betweentwo and fifty (2-50) amino acid residues linked by one or more covalentpeptide bond(s). The terms apply to naturally occurring amino acidpolymers as well as amino acid polymers in which one or more amino acidresidues is a non-naturally occurring amino acid (e.g., an amino acidanalog or non-natural amino acid).

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of greater than fifty (50) amino acid residues linkedby covalent peptide bonds. That is, a description directed to apolypeptide applies equally to a description of a protein, and viceversa. The terms apply to naturally occurring amino acid polymers aswell as amino acid polymers in which one or more amino acid residues isa non-naturally occurring amino acid (e.g., an amino acid analog). Asused herein, the terms encompass amino acid chains of any length,including full length proteins (e.g., antigens).

In the context of a peptide or polypeptide, the term “derivative” asused herein refers to a peptide or polypeptide that comprises an aminoacid sequence of the viral peptide or protein, or a fragment of a viralpeptide or protein, which has been altered by the introduction of aminoacid residue substitutions, deletions, or additions. The term“derivative” as used herein also refers to a viral peptide or protein,or a fragment of a viral peptide or protein, which has been chemicallymodified, e.g., by the covalent attachment of any type of molecule tothe polypeptide. For example, but not by way of limitation, a viralpeptide or protein or a fragment of the viral peptide or protein may bechemically modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, chemical cleavage, formulation, metabolicsynthesis of tunicamycin, linkage to a cellular ligand or other protein,etc. The derivatives are modified in a manner that is different fromnaturally occurring or starting peptide or polypeptides, either in thetype or location of the molecules attached. Derivatives further includedeletion of one or more chemical groups which are naturally present onthe viral peptide or protein. Further, a derivative of a viral peptideor protein or a fragment of a viral peptide or protein may contain oneor more non-classical amino acids. In specific embodiments, a derivativeis a functional derivative of the native or unmodified peptide orpolypeptide from which it was derived.

The term “functional derivative” refers to a derivative that retains oneor more functions or activities of the naturally occurring or startingpeptide or polypeptide from which it was derived. For example, afunctional derivative of a coronavirus S protein may retain the abilityto bind one or more of its receptors on a host cell. For example, afunctional derivative of a coronavirus N protein may retain the abilityto bind RNA or the package viral genome.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percent (%)amino acid sequence identity” with respect to a reference polypeptidesequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar,Inc.) software. Those skilled in the art can determine appropriateparameters for aligning sequences, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared.

A “modification” of an amino acid residue/position refers to a change ofa primary amino acid sequence as compared to a starting amino acidsequence, wherein the change results from a sequence alterationinvolving said amino acid residue/position. For example, typicalmodifications include substitution of the residue with another aminoacid (e.g., a conservative or non-conservative substitution), insertionof one or more (e.g., generally fewer than 5, 4, or 3) amino acidsadjacent to said residue/position, and/or deletion of saidresidue/position.

In the context of a peptide or polypeptide, the term “fragment” as usedherein refers to a peptide or polypeptide that comprises less than thefull length amino acid sequence. Such a fragment may arise, for example,from a truncation at the amino terminus, a truncation at the carboxyterminus, and/or an internal deletion of a residue(s) from the aminoacid sequence. Fragments may, for example, result from alternative RNAsplicing or from in vivo protease activity. In certain embodiments,fragments refers to polypeptides comprising an amino acid sequence of atleast 5 contiguous amino acid residues, at least 10 contiguous aminoacid residues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 30 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least 80 contiguous amino acid residues, atleast 90 contiguous amino acid residues, at least contiguous 100 aminoacid residues, at least 125 contiguous amino acid residues, at least 150contiguous amino acid residues, at least 175 contiguous amino acidresidues, at least 200 contiguous amino acid residues, at least 250, atleast 300, at least 350, at least 400, at least 450, at least 500, atleast 550, at least 600, at least 650, at least 700, at least 750, atleast 800, at least 850, at least 900, or at least 950 contiguous aminoacid residues of the amino acid sequence of a polypeptide. In a specificembodiment, a fragment of a polypeptide retains at least 1, at least 2,at least 3, or more functions of the polypeptide.

The term “immunogenic fragment” as used herein in the context of apeptide or polypeptide (e.g., a protein), refers to a fragment of apeptide or polypeptide that retains the ability of the peptide orpolypeptide in eliciting an immune response upon contacting the immunesystem of a mammal, including innate immune responses and/or adaptiveimmune responses. In some embodiments, an immunogenic fragment of apeptide or polypeptide can be an epitope.

The term “antigen” refers to a substance that can be recognized by theimmune system of a subject (including by the adaptive immune system),and is capable of triggering an immune response after the subject iscontacted with the antigen (including an antigen-specific immuneresponse). In certain embodiments, the antigen is a protein associatedwith a diseased cell, such as a cell infected by a pathogen or aneoplastic cell (e.g., tumor associated antigen (TAA)).

An “epitope” is the site on the surface of an antigen molecule to whicha single antibody molecule binds, such as a localized region on thesurface of an antigen that is capable of being bound to one or moreantigen binding regions of an antibody, and that has antigenic orimmunogenic activity in an animal, such as a mammal (e.g., a human),that is capable of eliciting an immune response. An epitope havingimmunogenic activity is a portion of a polypeptide that elicits anantibody response in an animal. An epitope having antigenic activity isa portion of a polypeptide to which an antibody binds as determined byany method well known in the art, including, for example, by animmunoassay. Antigenic epitopes need not necessarily be immunogenic.Epitopes often consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics. Antibody epitopes may be linear epitopes orconformational epitopes. Linear epitopes are formed by a continuoussequence of amino acids in a protein. Conformational epitopes are formedof amino acids that are discontinuous in the protein sequence, but whichare brought together upon folding of the protein into itsthree-dimensional structure. Induced epitopes are formed when the threedimensional structure of the protein is in an altered conformation, suchas following activation or binding of another protein or ligand. Incertain embodiments, an epitope is a three-dimensional surface featureof a polypeptide. In other embodiments, an epitope is linear feature ofa polypeptide. Generally an antigen has several or many differentepitopes and may react with many different antibodies.

The terms “severe acute respiratory syndrome coronavirus 2” or“SARS-CoV-2” or “2019-nCoV” are used interchangeably herein to refer tothe coronavirus that caused the pandemic of infectious diseases in 2019.GenBank™ accession number MN908947 provides exemplary genome sequence ofSARS-CoV-2 (SEQ ID NO:1).

The term “heterologous” refers an entity not found in nature to beassociated with (e.g., encoded by and/or expressed by the genome of) anaturally occurring coronavirus. The term “homologous” refers an entityfound in nature to be associated with (e.g., encoded by and/or expressedby the genome of) a naturally occurring coronavirus.

The term “genetic vaccine” as used herein refers to a therapeutic orprophylactic composition comprising at least one nucleic acid moleculeencoding an antigen associated with a target disease (e.g., aninfectious disease or a neoplastic disease). Administration of thevaccine to a subject (“vaccination”) allows for the production of theencoded peptide or protein, thereby eliciting an immune response againstthe target disease in the subject. In certain embodiments, the immuneresponse comprises adaptive immune response, such as the production ofantibodies against the encoded antigen, and/or activation andproliferations of immune cells capable of specifically eliminatingdiseased cells expressing the antigen. In certain embodiments, theimmune response further comprises innate immune response. According tothe present disclosure, a vaccine can be administered to a subjecteither before or after the onset of clinical symptoms of the targetdisease. In some embodiments, vaccination of a healthy or asymptomaticsubject renders the vaccinated subject immune or less susceptible to thedevelopment of the target disease. In some embodiments, vaccination of asubject showing symptoms of the disease improves the condition of, ortreats, the disease in the vaccinated subject.

The term “vector” refers to a substance that is used to carry or includea nucleic acid sequence, including for example, a nucleic acid sequenceencoding a viral peptide or protein as described herein, in order tointroduce a nucleic acid sequence into a host cell, or serve as atranscription template to carry out in vitro transcription reaction in acell-free system to produce mRNA. Vectors applicable for use include,for example, expression vectors, plasmids, phage vectors, viral vectors,episomes, and artificial chromosomes, which can include selectionsequences or markers operable for stable integration into a host cell'schromosome. Additionally, the vectors can include one or more selectablemarker genes and appropriate transcription or translation controlsequences. Selectable marker genes that can be included, for example,provide resistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Transcription or translation control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like, which are well known in the art. When two ormore nucleic acid molecules are to be co-transcribed or co-translated(e.g., nucleic acid molecules encoding two or more different viralpeptides or proteins), both nucleic acid molecules can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector transcription and/or translation, theencoding nucleic acids can be operationally linked to one commontranscription or translation control sequence or linked to differenttranscription or translation control sequences, such as one induciblepromoter and one constitutive promoter. The introduction of nucleic acidmolecules into a host cell can be confirmed using methods well known inthe art. Such methods include, for example, nucleic acid analysis suchas Northern blots or polymerase chain reaction (PCR) amplification ofmRNA, immunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the nucleic acid molecules are expressed in asufficient amount to produce a desired product (e.g., a mRNA transcriptof the nucleic acid as described herein), and it is further understoodthat expression levels can be optimized to obtain sufficient expressionusing methods well known in the art.

The terms “innate immune response” and “innate immunity” are recognizedin the art, and refer to non-specific defense mechanism a body's immunesystem initiates upon recognition of pathogen-associated molecularpatterns, which involves different forms of cellular activities,including cytokine production and cell death through various pathways.As used herein, innate immune responses include, without limitation,increased production of inflammation cytokines (e.g., type I interferonor IL-10 production), activation of the NFκB pathway, increasedproliferation, maturation, differentiation and/or survival of immunecells, and in some cases, induction of cell apoptosis. Activation of theinnate immunity can be detected using methods known in the art, such asmeasuring the (NF)-κB activation.

The terms “adaptive immune response” and “adaptive immunity” arerecognized in the art, and refer to antigen-specific defense mechanism abody's immune system initiates upon recognition of a specific antigen,which include both humoral response and cell-mediated responses. As usedherein, adaptive immune responses include cellular responses that istriggered and/or augmented by a vaccine composition, such as a geneticcomposition described herein. In some embodiments, the vaccinecomposition comprises an antigen that is the target of theantigen-specific adaptive immune response. In other embodiments, thevaccine composition, upon administration, allows the production in animmunized subject of an antigen that is the target of theantigen-specific adaptive immune response. Activation of an adaptiveimmune response can be detected using methods known in the art, such asmeasuring the antigen-specific antibody production, or the level ofantigen-specific cell-mediated cytotoxicity.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted immunoglobulin bound onto Fcreceptors (FcRs) present on certain cytotoxic cells (e.g., NaturalKiller (NK) cells, neutrophils, and macrophages) enable these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell with cytotoxins. The antibodies“arm” the cytotoxic cells and are absolutely required for such killing.NK cells, the primary cells for mediating ADCC, express FcγRIII only,whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression onhematopoietic cells is known (see, e.g., Ravetch and Kinet, 1991, Annu.Rev. Immunol. 9:457 92). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay (see, e.g., U.S. Pat. Nos. 5,500,362and 5,821,337) can be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively or additionally, ADCC activity of the moleculeof interest may be assessed in vivo, for example, in an animal model(see, e.g., Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-56).Antibodies with little or no ADCC activity may be selected for use.

“Antibody-dependent cellular phagocytosis” or “ADCP” refers to thedestruction of target cells via monocyte or macrophage-mediatedphagocytosis when immunoglobulin bound onto Fc receptors (FcRs) presenton certain phagocytotic cells (e.g., neutrophils, monocytes, andmacrophages) enable these phagocytotic cells to bind specifically to anantigen-bearing target cell and subsequently kill the target cell. Toassess ADCP activity of a molecule of interest, an in vitro ADCP assay(see, e.g., Bracher et al., 2007, J. Immunol. Methods 323:160-71) can beperformed. Useful phagocytotic cells for such assays include peripheralblood mononuclear cells (PBMC), purified monocytes from PBMC, or U937cells differentiated to the mononuclear type. Alternatively oradditionally, ADCP activity of the molecule of interest may be assessedin vivo, for example, in an animal model (see, e.g., Wallace et al.,2001, J. Immunol. Methods 248:167 82). Antibodies with little or no ADCPactivity may be selected for use.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. An exemplary FcR is a native sequence human FcR.Moreover, an exemplary FcR is one that binds an IgG antibody (e.g., agamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof (see, e.g., Daeron, 1997, Annu. Rev. Immunol. 15:203-34).Various FcRs are known (see, e.g., Ravetch and Kinet, 1991, Annu. Rev.Immunol. 9:457-92; Capel et al., 1994, Immunomethods 4:25-34; and deHaas et al., 1995, J. Lab. Clin. Med. 126:330-41). Other FcRs, includingthose to be identified in the future, are encompassed by the term “FcR”herein. The term also includes the neonatal receptor, FcRn, which isresponsible for the transfer of maternal IgGs to the fetus (see, e.g.,Guyer et al., 1976, J. Immunol. 117:587-93; and Kim et al., 1994, Eu. J.Immunol. 24:2429-34). Antibody variants with improved or diminishedbinding to FcRs have been described (see, e.g., WO 2000/42072; U.S. Pat.Nos. 7,183,387; 7,332,581; and 7.335,742; Shields et al. 2001, J. Biol.Chem. 9(2):6591-604).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay (see, e.g., Gazzano-Santoro et al., 1996, J.Immunol. Methods 202:163) may be performed. Polypeptide variants withaltered Fc region amino acid sequences (polypeptides with a variant Fcregion) and increased or decreased C1q binding capability have beendescribed (see, e.g., U.S. Pat. No. 6,194,551; WO 1999/51642; Idusogieet al., 2000, J. Immunol. 164: 4178-84). Antibodies with little or noCDC activity may be selected for use.

The term “antibody” is intended to include a polypeptide product of Bcells within the immunoglobulin class of polypeptides that is able tobind to a specific molecular antigen and is composed of two identicalpairs of polypeptide chains, wherein each pair has one heavy chain(about 50-70 kDa) and one light chain (about 25 kDa), eachamino-terminal portion of each chain includes a variable region of about100 to about 130 or more amino acids, and each carboxy-terminal portionof each chain includes a constant region. See, e.g., AntibodyEngineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed.1997). In specific embodiments, the specific molecular antigen can bebound by an antibody provided herein, including a polypeptide, afragment or an epitope thereof. Antibodies also include, but are notlimited to, synthetic antibodies, recombinantly produced antibodies,camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies,and functional fragments of any of the above, which refers to a portionof an antibody heavy or light chain polypeptide that retains some or allof the binding activity of the antibody from which the fragment wasderived. Non-limiting examples of functional fragments includesingle-chain Fvs (scFv) (e.g., including monospecific, bispecific,etc.), Fab fragments, F(ab′) fragments, F(ab)₂ fragments, F(ab′)₂fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments,diabody, triabody, tetrabody, and minibody. In particular, antibodiesprovided herein include immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, for example,antigen-binding domains or molecules that contain an antigen-bindingsite (e.g., one or more CDRs of an antibody). Such antibody fragmentscan be found in, for example, Harlow and Lane, Antibodies: A LaboratoryManual (1989); Mol. Biology and Biotechnology: A Comprehensive DeskReference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497-515; andDay, Advanced Immunochemistry (2d ed. 1990). The antibodies providedherein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or anysubclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) ofimmunoglobulin molecule.

The term “administer” or “administration” refers to the act of injectingor otherwise physically delivering a substance as it exists outside thebody (e.g., a lipid nanoparticle composition as described herein) into apatient, such as by mucosal, intradermal, intravenous, intramusculardelivery, and/or any other method of physical delivery described hereinor known in the art. When a disease, disorder, condition, or a symptomthereof, is being treated, administration of the substance typicallyoccurs after the onset of the disease, disorder, condition, or symptomsthereof. When a disease, disorder, condition, or symptoms thereof, arebeing prevented, administration of the substance typically occurs beforethe onset of the disease, disorder, condition, or symptoms thereof.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode (e.g., for a period of time such as days, weeks, months,or years) as opposed to an acute mode, so as to maintain the initialtherapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

The term “targeted delivery” or the verb form “target” as used hereinrefers to the process that promotes the arrival of a delivered agent(such as a therapeutic payload molecule in a lipid nanoparticlecomposition as described herein) at a specific organ, tissue, celland/or intracellular compartment (referred to as the targeted location)more than any other organ, tissue, cell or intracellular compartment(referred to as the non-target location). Targeted delivery can bedetected using methods known in the art, for example, by comparing theconcentration of the delivered agent in a targeted cell population withthe concentration of the delivered agent at a non-target cell populationafter systemic administration. In certain embodiments, targeted deliveryresults in at least 2 fold higher concentration at a targeted locationas compared to a non-target location.

An “effective amount” is generally an amount sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with a disease, disorder, or condition, including,for example, infection and neoplasia. In some embodiments, the effectiveamount is a therapeutically effective amount or a prophylacticallyeffective amount.

The term “therapeutically effective amount” as used herein refers to theamount of an agent (e.g., a vaccine composition) that is sufficient toreduce and/or ameliorate the severity and/or duration of a givendisease, disorder, or condition, and/or a symptom related thereto (e.g.,an infectious disease such as caused by viral infection, or a neoplasticdisease such as cancer). A “therapeutically effective amount” of asubstance/molecule/agent of the present disclosure (e.g., the lipidnanoparticle composition as described herein) may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the substance/molecule/agent to elicit adesired response in the individual. A therapeutically effective amountencompasses an amount in which any toxic or detrimental effects of thesubstance/molecule/agent are outweighed by the therapeuticallybeneficial effects. In certain embodiments, the term “therapeuticallyeffective amount” refers to an amount of a lipid nanoparticlecomposition as described herein or a therapeutic or prophylactic agentcontained therein (e.g., a therapeutic mRNA) effective to “treat” adisease, disorder, or condition, in a subject or mammal.

A “prophylactically effective amount” is an amount of a pharmaceuticalcomposition that, when administered to a subject, will have the intendedprophylactic effect, e.g., preventing, delaying, or reducing thelikelihood of the onset (or reoccurrence) of a disease, disorder,condition, or associated symptom(s) (e.g., an infectious disease such ascaused by viral infection, or a neoplastic disease such as cancer).Typically, but not necessarily, since a prophylactic dose is used insubjects prior to or at an earlier stage of a disease, disorder, orcondition, a prophylactically effective amount may be less than atherapeutically effective amount. The full therapeutic or prophylacticeffect does not necessarily occur by administration of one dose, and mayoccur only after administration of a series of doses. Thus, atherapeutically or prophylactically effective amount may be administeredin one or more administrations.

The terms “prevent,” “preventing,” and “prevention” refer to reducingthe likelihood of the onset (or recurrence) of a disease, disorder,condition, or associated symptom(s) (e.g., an infectious disease such ascaused by viral infection, or a neoplastic disease such as cancer).

The terms “manage,” “managing,” and “management” refer to the beneficialeffects that a subject derives from a therapy (e.g., a prophylactic ortherapeutic agent), which does not result in a cure of the disease. Incertain embodiments, a subject is administered one or more therapies(e.g., prophylactic or therapeutic agents, such as a lipid nanoparticlecomposition as described herein) to “manage” an infectious or neoplasticdisease, one or more symptoms thereof, so as to prevent the progressionor worsening of the disease.

The term “prophylactic agent” refers to any agent that can totally orpartially inhibit the development, recurrence, onset, or spread ofdisease and/or symptom related thereto in a subject.

The term “therapeutic agent” refers to any agent that can be used intreating, preventing, or alleviating a disease, disorder, or condition,including in the treatment, prevention, or alleviation of one or moresymptoms of a disease, disorder, or condition and/or a symptom relatedthereto.

The term “therapy” refers to any protocol, method, and/or agent that canbe used in the prevention, management, treatment, and/or amelioration ofa disease, disorder, or condition. In certain embodiments, the terms“therapies” and “therapy” refer to a biological therapy, supportivetherapy, and/or other therapies useful in the prevention, management,treatment, and/or amelioration of a disease, disorder, or condition,known to one of skill in the art such as medical personnel.

As used herein, a “prophylactically effective serum titer” is the serumtiter of an antibody in a subject (e.g., a human), that totally orpartially inhibits the development, recurrence, onset, or spread of adisease, disorder, or condition, and/or symptom related thereto in thesubject.

In certain embodiments, a “therapeutically effective serum titer” is theserum titer of an antibody in a subject (e.g., a human), that reducesthe severity, the duration, and/or the symptoms associated with adisease, disorder, or condition, in the subject.

The term “serum titer” refers to an average serum titer in a subjectfrom multiple samples (e.g., at multiple time points) or in a populationof at least 10, at least 20, at least 40 subjects, up to about 100,1000, or more.

The term “side effects” encompasses unwanted and/or adverse effects of atherapy (e.g., a prophylactic or therapeutic agent). Unwanted effectsare not necessarily adverse. An adverse effect from a therapy (e.g., aprophylactic or therapeutic agent) might be harmful, uncomfortable, orrisky. Examples of side effects include, diarrhea, cough,gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominalcramping, fever, pain, loss of body weight, dehydration, alopecia,dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects,fatigue, dry mouth, loss of appetite, rashes or swellings at the site ofadministration, flu-like symptoms such as fever, chills, and fatigue,digestive tract problems, and allergic reactions. Additional undesiredeffects experienced by patients are numerous and known in the art. Manyare described in Physician's Desk Reference (68th ed. 2014).

The terms “subject” and “patient” may be used interchangeably. As usedherein, in certain embodiments, a subject is a mammal, such as anon-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate(e.g., monkey and human). In specific embodiments, the subject is ahuman. In one embodiment, the subject is a mammal (e.g., a human) havingan infectious disease or neoplastic disease. In another embodiment, thesubject is a mammal (e.g., a human) at risk of developing an infectiousdisease or neoplastic disease.

The term “elderly human” refers to a human 65 years or older. The term“human adult” refers to a human that is 18 years or older. The term“human child” refers to a human that is 1 year to 18 years old. The term“human toddler” refers to a human that is 1 year to 3 years old. Theterm “human infant” refers to a newborn to 1 year old year human.

The term “detectable probe” refers to a composition that provides adetectable signal. The term includes, without limitation, anyfluorophore, chromophore, radiolabel, enzyme, antibody or antibodyfragment, and the like, that provide a detectable signal via itsactivity.

The term “detectable agent” refers to a substance that can be used toascertain the existence or presence of a desired molecule, such as anantigen encoded by an mRNA molecule as described herein, in a sample orsubject. A detectable agent can be a substance that is capable of beingvisualized or a substance that is otherwise able to be determined and/ormeasured (e.g., by quantitation).

“Substantially all” refers to at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or about 100%.

As used herein, and unless otherwise indicated, the term “about” or“approximately” means an acceptable error for a particular value asdetermined by one of ordinary skill in the art, which depends in part onhow the value is measured or determined. In certain embodiments, theterm “about” or “approximately” means within 1, 2, 3, or 4 standarddeviations. In certain embodiments, the term “about” or “approximately”means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.05%, or less of a given value or range.

The singular terms “a,” “an,” and “the” as used herein include theplural reference unless the context clearly indicates otherwise.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference in their entirety as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedcan be different from the actual publication dates which can need to beindependently confirmed.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the descriptions in the Experimental section and examplesare intended to illustrate but not limit the scope of inventiondescribed in the claims.

6.3 Therapeutic Nucleic Acids

In one aspect, provided herein are therapeutic nucleic acid moleculesfor the management, prevention and treatment of coronavirus infection.In some embodiments, the therapeutic nucleic acid encodes a peptide orpolypeptide, which upon administration into a subject in need thereof,is expressed by the cells in the subject to produce the encoded peptideor polypeptide. In some embodiments, the therapeutic nucleic acidmolecules are DNA molecules. In other embodiments, the therapeuticnucleic acid molecules are RNA molecules. In particular embodiments, thetherapeutic nucleic acid molecules are mRNA molecules.

In some embodiments, the therapeutic nucleic acid molecule is formulatedin a vaccine composition. In some embodiments, the vaccine compositionis a genetic vaccine as described herein. In some embodiments, thevaccine composition comprises an mRNA molecule as described herein.

In some embodiments, the mRNA molecule of the present disclosure encodesa peptide or polypeptide of interest, including any naturally ornon-naturally occurring or otherwise modified polypeptide. A peptide orpolypeptide encoded by an mRNA may be of any size and may have anysecondary structure or activity. In some embodiments, the polypeptideencoded by an mRNA payload can have a therapeutic effect when expressedin a cell.

In some embodiment, the mRNA molecule of the present disclosurecomprises at least one coding region encoding a peptide or polypeptideof interest (e.g., an open reading frame (ORF)). In some embodiments,the nucleic acid molecule further comprises at least one untranslatedregion (UTR). In particular embodiments, the untranslated region (UTR)is located upstream (to the 5′-end) of the coding region, and isreferred to herein as the 5′-UTR. In particular embodiments, theuntranslated region (UTR) is located downstream (to the 3′-end) of thecoding region, and is referred to herein as the 3′-UTR. In particularembodiments, the nucleic acid molecule comprises both a 5′-UTR and a3′-UTR. In some embodiments, the 5′-UTR comprises a 5′-Cap structure. Insome embodiments, the nucleic acid molecule comprises a Kozak sequence(e.g., in the 5′-UTR). In some embodiments, the nucleic acid moleculecomprises a poly-A region (e.g., in the 3′-UTR). In some embodiments,the nucleic acid molecule comprises a polyadenylation signal (e.g., inthe 3′-UTR). In some embodiments, the nucleic acid molecule comprisesstabilizing region (e.g., in the 3′-UTR). In some embodiments, thenucleic acid molecule comprises a secondary structure. In someembodiments, the secondary structure is a stem-loop. In someembodiments, the nucleic acid molecule comprises a stem-loop sequence(e.g., in the 5′-UTR and/or the 3′-UTR). In some embodiments, thenucleic acid molecule comprises one or more intronic regions capable ofbeing excised during splicing. In a specific embodiment, the nucleicacid molecule comprises one or more region selected from a 5′-UTR, and acoding region. In a specific embodiment, the nucleic acid moleculecomprises one or more region selected from a coding region and a 3′-UTR.In a specific embodiment, the nucleic acid molecule comprises one ormore region selected from a 5′-UTR, a coding region, and a 3′-UTR.

6.3.1 Coding Region

In some embodiments, the nucleic acid molecule of the present disclosurecomprises at least one coding region. In some embodiments, the codingregion is an open reading frame (ORF) that encodes for a single peptideor protein. In some embodiments, the coding region comprises at leasttwo ORFs, each encoding a peptide or protein. In those embodiments wherethe coding region comprises more than one ORFs, the encoded peptidesand/or proteins can be the same as or different from each other. In someembodiments, the multiple ORFs in a coding region are separated bynon-coding sequences. In specific embodiments, a non-coding sequenceseparating two ORFs comprises an internal ribosome entry sites (IRES).

Without being bound by the theory, it is contemplated that an internalribosome entry sites (IRES) can act as the sole ribosome binding site,or serve as one of multiple ribosome binding sites of an mRNA. An mRNAmolecule containing more than one functional ribosome binding site canencode several peptides or proteins that are translated independently bythe ribosomes (e.g., multicistronic mRNA). Accordingly, in someembodiments, the nucleic acid molecule of the present disclosure (e.g.,mRNA) comprises one or more internal ribosome entry sites (IRES).Examples of IRES sequences that can be used in connection with thepresent disclosure include, without limitation, those from picomaviruses(e.g., FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (SIV) orcricket paralysis viruses (CrPV).

In various embodiments, the nucleic acid molecule of the presentdisclosure encodes for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morethan 10 peptides or proteins. Peptides and proteins encoded by a nucleicacid molecule can be the same or different. In some embodiments, thenucleic acid molecule of the present disclosure encodes a dipeptide(e.g., camosine and anserine). In some embodiments, the nucleic acidmolecule encodes a tripeptide. In some embodiments, the nucleic acidmolecule encodes a tetrapeptide. In some embodiments, the nucleic acidmolecule encodes a pentapeptide. In some embodiments, the nucleic acidmolecule encodes a hexapeptide. In some embodiments, the nucleic acidmolecule encodes a heptapeptide. In some embodiments, the nucleic acidmolecule encodes an octapeptide. In some embodiments, the nucleic acidmolecule encodes a nonapeptide. In some embodiments, the nucleic acidmolecule encodes a decapeptide. In some embodiments, the nucleic acidmolecule encodes a peptide or polypeptide that has at least about 15amino acids. In some embodiments, the nucleic acid molecule encodes apeptide or polypeptide that has at least about 50 amino acids. In someembodiments, the nucleic acid molecule encodes a peptide or polypeptidethat has at least about 100 amino acids. In some embodiments, thenucleic acid molecule encodes a peptide or polypeptide that has at leastabout 150 amino acids. In some embodiments, the nucleic acid moleculeencodes a peptide or polypeptide that has at least about 300 aminoacids. In some embodiments, the nucleic acid molecule encodes a peptideor polypeptide that has at least about 500 amino acids. In someembodiments, the nucleic acid molecule encodes a peptide or polypeptidethat has at least about 1000 amino acids.

In some embodiments, the nucleic acid molecule of the present disclosureis at least about 30 nucleotides (nt) in length. In some embodiments,the nucleic acid molecule is at least about 35 nt in length. In someembodiments, the nucleic acid molecule is at least about 40 nt inlength. In some embodiments, the nucleic acid molecule is at least about45 nt in length. In some embodiments the nucleic acid molecule is atleast about 50 nt in length. In some embodiments, the nucleic acidmolecule is at least about 55 nt in length. In some embodiments, thenucleic acid molecule is at least about 60 nt in length. In someembodiments, the nucleic acid molecule is at least about 65 nt inlength. In some embodiments, the nucleic acid molecule is at least about70 nt in length. In some embodiments, the nucleic acid molecule is atleast about 75 nt in length. In some embodiments, the nucleic acidmolecule is at least about 80 nt in length. In some embodiments thenucleic acid molecule is at least about 85 nt in length. In someembodiments, the nucleic acid molecule is at least about 90 nt inlength. In some embodiments, the nucleic acid molecule is at least about95 nt in length. In some embodiments, the nucleic acid molecule is atleast about 100 nt in length. In some embodiments, the nucleic acidmolecule is at least about 120 nt in length. In some embodiments, thenucleic acid molecule is at least about 140 nt in length. In someembodiments, the nucleic acid molecule is at least about 160 nt inlength. In some embodiments, the nucleic acid molecule is at least about180 nt in length. In some embodiments, the nucleic acid molecule is atleast about 200 nt in length. In some embodiments, the nucleic acidmolecule is at least about 250 nt in length. In some embodiments, thenucleic acid molecule is at least about 300 nt in length. In someembodiments, the nucleic acid molecule is at least about 400 nt inlength. In some embodiments, the nucleic acid molecule is at least about500 nt in length. In some embodiments, the nucleic acid molecule is atleast about 600 nt in length. In some embodiments, the nucleic acidmolecule is at least about 700 nt in length. In some embodiments, thenucleic acid molecule is at least about 800 nt in length. In someembodiments, the nucleic acid molecule is at least about 900 nt inlength. In some embodiments, the nucleic acid molecule is at least about1000 nt in length. In some embodiments, the nucleic acid molecule is atleast about 1100 nt in length. In some embodiments, the nucleic acidmolecule is at least about 1200 nt in length. In some embodiments, thenucleic acid molecule is at least about 1300 nt in length. In someembodiments, the nucleic acid molecule is at least about 1400 nt inlength. In some embodiments, the nucleic acid molecule is at least about1500 nt in length. In some embodiments, the nucleic acid molecule is atleast about 1600 nt in length. In some embodiments, the nucleic acidmolecule is at least about 1700 nt in length. In some embodiments, thenucleic acid molecule is at least about 1800 nt in length. In someembodiments, the nucleic acid molecule is at least about 1900 nt inlength. In some embodiments, the nucleic acid molecule is at least about2000 nt in length. In some embodiments, the nucleic acid molecule is atleast about 2500 nt in length. In some embodiments, the nucleic acidmolecule is at least about 3000 nt in length. In some embodiments, thenucleic acid molecule is at least about 3500 nt in length. In someembodiments, the nucleic acid molecule is at least about 4000 nt inlength. In some embodiments, the nucleic acid molecule is at least about4500 nt in length. In some embodiments, the nucleic acid molecule is atleast about 5000 nt in length.

In specific embodiments, the therapeutic nucleic acid of the presentdisclosure are formulated as a vaccine composition (e.g., a geneticvaccine) as described herein. In some embodiments, the therapeuticnucleic acid encodes a peptide or protein capable of eliciting immunityagainst one or more target conditions or disease. In some embodiments,the target condition is related to or caused by infection by a pathogen,such as a coronavirus (e.g. COVID-19), influenza, measles, humanpapillomavirus (HPV), rabies, meningitis, whooping cough, tetanus,plague, hepatitis, and tuberculosis. In some embodiments, thetherapeutic nucleic acid sequence (e.g., mRNA) encoding a pathogenicprotein characteristic for the pathogen, or an immunogenic fragment(e.g., epitope) or derivative thereof. The vaccine, upon administrationto a vaccinated subject, allows for expression of the encoded pathogenicprotein (or the immunogenic fragment or derivative thereof), therebyeliciting immunity in the subject against the pathogen.

In specific embodiments, provided herein are therapeutic compositions(e.g., vaccine compositions) for the management, prevention andtreatment of infectious diseases or conditions caused by coronaviruses.Coronaviruses belong to the family Coronaviridae in the orderNidoviralesn and are classified into four genera: Alphacoronavirus,Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Among them,alpha- and betacoronaviruses infect mammals, gammacoronaviruses infectavian species, and deltacoronaviruses infect both mammalian and avianspecies. Representative alphacoronaviruses include human coronavirusNL63 (HCoV-NL63), porcine transmissible gastroenteritis coronavirus(TGEV), PEDV, and porcine respiratory coronavirus (PRCV). Representativebetacoronaviruses include SARS-CoV, MERS-CoV, bat coronavirus HKU4,mouse hepatitis coronavirus (MHV), bovine coronavirus (BCoV), and humancoronavirus OC43. Representative gamma- and deltacoronaviruses includeavian infectious bronchitis coronavirus (IBV) and porcinedeltacoronavirus (PdCV), respectively. Li et al. Annu Rev Virol. 20163(1):237-261.

Without being bound by the theory, it is contemplated that coronavirusesare enveloped, positive-stranded RNA viruses. They have large genomes,typically ranging from 27 to 32 kb. The genome is packed inside ahelical capsid formed by the nucleocapsid (N) protein and furthersurrounded by an envelope. Associated with the viral envelope are atleast three structural proteins: the membrane (M) protein and theenvelope (E) protein are involved in virus assembly, whereas the spike(S) protein mediates virus entry into host cells. Some coronavirusesalso encode an envelope-associated hemagglutinin-esterase (HE) protein.Among these structural proteins, the spike forms large protrusions fromthe virus surface, giving coronaviruses the appearance of having crowns.It is further contemplated that in addition to mediating virus entry,the spike protein may play a role in determining the viral host rangeand tissue tropism and a major inducer of host immune responses. Li etal. Annu Rev Virol. 2016 3(1):237-261.

Accordingly, in some embodiments, provided herein are therapeuticnucleic acids encoding a viral peptide or protein derived from acoronavirus. In some embodiments, the nucleic acid encodes a viralpeptide or protein derived from a coronavirus, where the viral peptideor protein is one or more selected from (a) the N protein, (b) the Mprotein, (c) the E protein, (d) the S protein, (e) the HE protein, (f)an immunogenic fragment of any one of (a) to (e), and (g) a functionalderivative of any one of (a) to (f).

Without being bound by the theory, it is contemplated that thecoronavirus S protein contains three segments: an ectodomain, asingle-pass transmembrane anchor, and an intracellular tail. It isfurther contemplated that the ectodomain comprises a receptor-bindingsubunit 51 and a membrane-fusion subunit S2. The 51 subunit furthercomprises two major domains: the N-terminal domain (S1-NTD) andC-terminal domain (S1-CTD). It is further contemplated that one or bothof these domains in the 51 subunit may bind to receptors on a host cell,and function as the receptor binding domain (RBD). Particularly, it isfurther contemplated that host receptors recognized by either domains inthe 51 subunit include angiotensin-converting enzyme 2 (ACE2),aminopeptidase N (APN), dipeptidyl peptidase 4 (DPP4), carcinoembryonicantigen-related cell adhesion molecule 1 (CEACAM1) and sugar. It isfurther contemplated that S1-CTD contains two subdomains: a corestructure and a receptor-binding motif (RBM). The RBM binds to ACE2receptors on host cells.

Accordingly, in some embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the coronavirus S protein, or an immunogenicfragment of the S protein, or a functional derivative of the S proteinor the immunogenic fragment thereof. In specific embodiments, theimmunogenic fragment of the S protein is selected from the ectodomain,the S1 subunit, the receptor binding domain (RBD), and the receptorbinding motif (RBM). In other embodiments, the immunogenic fragment ofthe S protein is selected from the transmembrane domain, theintracellular tail, the S2 subunit, the S1-NTD domain, and the S1-CTDdomain. Table 1 shows exemplary SARS-CoV-2 native antigen sequences.

TABLE 1 Exemplary native sequences of SARS-CoV-2 antigens. SEQUENCENAME (SEQ ID NO:) AMINO ACID OR NUCLEIC ACID SEQUENCE SARS-CoV-2MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS spike protein withVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVY native signalFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCND peptide amino acidPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGK sequence (SEQ IDQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDL NO: 2)PIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT SARS-CoV-2ATGTTTGTTTTTCTTGTTTTATTGCCATTAGTCTCTAGTCAGTGT spike protein withGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAA native signalTTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGAT peptide codingCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTT sequence (SEQ IDCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAAT NO: 3)GGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAA ATTACATTACACA SARS-CoV-2QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF spike proteinSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWI ectodomain (ECD)FGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK amino acidSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVF sequence (SEQ IDKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL NO: 4)ALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE LGKYEQYIK SARS-CoV-2CAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATA spike protein ECDCACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTT coding sequenceTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTT (SEQ ID NO: 5)TCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAACTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATAT AAAA SARS-CoV-2QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF spike protein S1SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWI subunit amino acidFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK sequence (SEQ IDSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVF NO: 6)KNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR SARS-CoV-2CAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATAC spike protein S1ACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTC subunit codingAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTC sequence (SEQ IDTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACC NO: 7)AATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGT SARS-CoV-2RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD spike proteinYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI receptor bindingAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYR domain (RBD)LFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT N spanning positionsGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF 319-541 (RBD-1) amino acidsequence (SEQ ID NO: 8) SARS-CoV-2AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACA spike proteinAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGC RBD-1 codingATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTG sequence (SEQ IDCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAG NO: 9)TGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAACTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTC SARS-CoV-2NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS spike protein RBDTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD spanning positionsYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPF 331-529 (RBD-2)ERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV amino acidVVLSFELLHAPATVCGPKK sequence (SEQ ID NO: 10) SARS-CoV-2AATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACC spike proteinAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAA RBD-2 codingCTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCC sequence (SEQ IDACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTC NO: 11)TGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAACTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTG TGGACCTAAAAAG SARS-CoV-2NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS spike protein RBDTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD spanning positionsYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPF 331-524 (RBD-3)ERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV amino acid VVLSFELLHAPATVsequence (SEQ ID NO: 12) SARS-CoV-2AATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCAC spike proteinCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGC RBD-3 codingAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTT sequence (SEQ IDTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGA NO: 13)TCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAACTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCA CCAGCAACTGTT SARS-CoV-2RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD spike protein RBDYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI spanning positionsAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYR 319-529 (RBD-4)LFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN amino acidGVGYQPYRVVVLSFELLHAPATVCGPKK sequence (SEQ ID NO: 14) SARS-CoV-2AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACA spike proteinAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGC RBD-4 codingATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTG sequence (SEQ IDCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAG NO: 15)TGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAACTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAA AG SARS-CoV-2VGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF spike protein PLQSYGFQPTreceptor binding motif (RBM) amino acid sequence (SEQ ID NO: 16)SARS-CoV-2 GTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAspike protein RBM ATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGcoding sequence CCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACT(SEQ ID NO: 17) TTCCTTTACAATCATATGGTTTCCAACCCACT SARS-CoV-2MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGL nucleocapsidPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRA protein amino acidTRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVA sequence (SEQ IDTEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGG NO: 18)SQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDD FSKQLQQSMSSADSTQASARS-CoV-2 ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCA nucleocapsidTTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAA protein codingTGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCA sequence (SEQ IDAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTC NO: 19)AACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGCTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCC

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the S protein of coronavirus SARS-CoV-2, wherein theS protein has an amino acid sequence of SEQ ID NO:2. In particularembodiments, the therapeutic nucleic acid of the present disclosureencodes the S protein of coronavirus SARS-CoV-2, and wherein thetherapeutic nucleic acid comprises a DNA coding sequence of SEQ ID NO:3.In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the S protein of coronavirus SARS-CoV-2, and whereinthe therapeutic nucleic acid comprises a RNA sequence transcribed fromthe DNA coding sequence of SEQ ID NO:3. In particular embodiments, thenucleic acid molecule is an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the ectodomain (ECD) of the S protein of coronavirusSARS-CoV-2, and wherein the ectodomain has an amino acid sequence SEQ IDNO:4. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the ECD of the S protein of coronavirusSARS-CoV-2, and wherein the therapeutic nucleic acid comprises a DNAcoding sequence of SEQ ID NO:5. In particular embodiments, thetherapeutic nucleic acid of the present disclosure encodes the ECD ofthe S protein of coronavirus SARS-CoV-2, and wherein the therapeuticnucleic acid comprises a RNA sequence transcribed from the DNA codingsequence of SEQ ID NO:5. In some embodiments, the RNA sequence is invitro transcribed. In particular embodiments, the nucleic acid moleculeis an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the S1 subunit of the S protein of coronavirusSARS-CoV-2, and wherein the S1 subunit has an amino acid sequence of SEQID NO:6. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the S1 subunit of the S protein ofcoronavirus SARS-CoV-2, and wherein the therapeutic nucleic acidcomprises a DNA coding sequence SEQ ID NO:7. In particular embodiments,the therapeutic nucleic acid of the present disclosure encodes the S1subunit of the S protein of coronavirus SARS-CoV-2, and wherein thetherapeutic nucleic acid comprises a RNA sequence transcribed from theDNA coding sequence of SEQ ID NO:7. In some embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, the nucleicacid molecule is an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes an immunogenic fragment of the S protein ofcoronavirus SARS-CoV-2. In some embodiments, the immunogenic fragment isthe receptor binding domain (RBD) of the S protein of coronavirusSARS-CoV-2. In some embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the RBD sequence locates at residues 319-541of the S protein, and has an amino acid sequence of SEQ ID NO:8. Inparticular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the RBD sequence of the S protein of coronavirusSARS-CoV-2, and wherein the therapeutic nucleic acid comprises a DNAcoding sequence of SEQ ID NO:9. In particular embodiments, thetherapeutic nucleic acid of the present disclosure encodes the RBDsequence of the S protein of coronavirus SARS-CoV-2, and wherein thetherapeutic nucleic acid comprises a RNA sequence transcribed from theDNA coding sequence of SEQ ID NO:9. In some embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, the nucleicacid molecule is an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the RBD sequence located at residues 331-529 of the Sprotein of coronavirus SARS-CoV-2, and has an amino acid sequence of SEQID NO:10. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the RBD sequence of the S protein ofcoronavirus SARS-CoV-2, and wherein the therapeutic nucleic acidcomprises a DNA coding sequence of SEQ ID NO:11. In particularembodiments, the therapeutic nucleic acid of the present disclosureencodes the RBD sequence of the S protein of coronavirus SARS-CoV-2, andwherein the therapeutic nucleic acid comprises a RNA sequencetranscribed from the DNA coding sequence of SEQ ID NO:11. In someembodiments, the RNA sequence is in vitro transcribed. In particularembodiments, the nucleic acid molecule is an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes a RBD sequence of the S protein of coronavirusSARS-CoV-2, and wherein the RBD sequence locates at residues 331-524 ofthe S protein, and has an amino acid sequence of SEQ ID NO:12. Inparticular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes a RBD sequence of the S protein of coronavirusSARS-CoV-2, and wherein the therapeutic nucleic acid comprises a DNAcoding sequence of SEQ ID NO:13. In particular embodiments, thetherapeutic nucleic acid of the present disclosure encodes a RBDsequence of the S protein of coronavirus SARS-CoV-2, and wherein thetherapeutic nucleic acid comprises a RNA sequence transcribed from theDNA coding sequence of SEQ ID NO:13. In some embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, the nucleicacid molecule is an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes a RBD sequence of the S protein of coronavirusSARS-CoV-2, and wherein the RBD domain locates at residues 319-529 ofthe S protein, and has an amino acid sequence of SEQ ID NO:14. Inparticular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes a RBD sequence of the S protein of coronavirusSARS-CoV-2, and wherein the therapeutic nucleic acid comprises a DNAcoding sequence of SEQ ID NO:15. In particular embodiments, thetherapeutic nucleic acid of the present disclosure encodes a RBDsequence of the S protein of coronavirus SARS-CoV-2, and wherein thetherapeutic nucleic acid comprises a RNA sequence transcribed from theDNA coding sequence of SEQ ID NO:15. In some embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, the nucleicacid molecule is an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the receptor binding motif (RBM) sequence of the Sprotein of coronavirus SARS-CoV-2, and wherein the RBM has an amino acidsequence of SEQ ID NO:16. In particular embodiments, the therapeuticnucleic acid of the present disclosure encodes the RBM of the S proteinof coronavirus SARS-CoV-2, and wherein the therapeutic nucleic acidcomprises a DNA coding sequence of SEQ ID NO:17. In particularembodiments, the therapeutic nucleic acid of the present disclosureencodes the RBM of the S protein of coronavirus SARS-CoV-2, and whereinthe therapeutic nucleic acid comprises a RNA sequence transcribed fromthe DNA coding sequence of SEQ ID NO:17. In some embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, the nucleicacid molecule is an mRNA molecule.

In some embodiments, the therapeutic nucleic acid of the presentdisclosure encodes a functional derivative of RBD. In particularembodiments, the functional derivative of RBD comprises one or moremutations that increase binding affinity of the RBD to host receptors ascompared to RBD without such mutations. In particular embodiments, thecoronavirus is SARS-CoV, and wherein the mutation is K479N and/or S487T.

In particular embodiments, the coronavirus is SARS-CoV-2, and whereinthe mutation is N501 T. Table 2 shows exemplary sequences of S proteinof coronavirus SARS-CoV-2 or antigenic fragments thereof having theN501T mutation.

TABLE 2 Exemplary mutated sequences of SARS-CoV-2 antigens. SEQUENCENAME (SEQ ID NO:) AMINO ACID OR NUCLEIC ACID SEQUENCE SARS-CoV-2MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS spike proteinVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYF withASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCND native signalPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG peptide and anNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLP

IGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFL

LKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQP amino acidTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLY sequenceNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT (SEQ ID NO: 20)GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT

GVGY QPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTlEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT SARS-CoV-2QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF spike protein ECDSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWI with an 

FGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKS

WMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI aminoDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLA acid sequenceLHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA (SEQ ID NO: 21)VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT

GVGYQPYRVVVLS FELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE LGKYEQYIK SARS-CoV-2QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF spike protein S1SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWI subunit with anFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNK

SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN

IDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL aminoALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA acid sequenceVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITN (SEQ ID NO: 22)LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT

GVGYQPYRVVVL SFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR SARS-CoV-2RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD spike proteinYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI RBD-1 with anAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF

RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT

GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (RBD-5) amino acid sequence(SEQ ID NO: 23) SARS-CoV-2ITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF spike proteinKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYN RBD-2 with anYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER

DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT

GVGYQPYR

VVVLSFELLHAPATVCGPKK (RBD-6) amino acid sequence (SEQ ID NO: 24)SARS-CoV-2 NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS spike proteinTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD RBD-3 with anYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFE

RDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT

GVGYQPYR

VVVLSFELLHAPATV (RBD-7) amino acid sequence (SEQ ID NO: 25) SARS-CoV-2RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD spike proteinYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI RBD-4 with anAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF

RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT

GVGYQPYRVVVLSFELLHAPATVCGPKK (RBD-8) amino acid sequence (SEQ ID NO: 26)SARS-CoV-2 AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACA spike proteinAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCA RBD-8 codingTCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCT sequence (SEQ IDGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAG NO: 27)TGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAACTCTAAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTACTGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAA AAG

In particular embodiments, the therapeutic nucleic acid encodes afunctional derivative of the S protein of coronavirus SARS-CoV-2. Inparticular embodiments, the encoded functional derivative of the Sprotein comprises an amino acid substitution N501T. In particularembodiments, the encoded functional derivative of the S proteincomprises an amino acid sequence of SEQ ID NO: 20.

In particular embodiments, the therapeutic nucleic acid encodes afunctional derivative of the ectodomain of the S protein of coronavirusSARS-CoV-2. In particular embodiments, the encoded functional derivativeof the S protein ectodomain comprises an amino acid substitution N501 T.In particular embodiments, the encoded functional derivative of the Sprotein ectodomain comprises an amino acid sequence of SEQ ID NO: 21.

In particular embodiments, the therapeutic nucleic acid encodes afunctional derivative of the 51 subunit the S protein of coronavirusSARS-CoV-2. In particular embodiments, the encoded functional derivativeof the S protein 51 subunit comprises an amino acid substitution N501 T.In particular embodiments, the encoded functional derivative of the Sprotein 51 subunit comprises an amino acid sequence of SEQ ID NO: 22.

In particular embodiments, the therapeutic nucleic acid encodes afunctional derivative of the receptor binding domain (RBD) sequence ofthe S protein of coronavirus SARS-CoV-2. In particular embodiments, theencoded functional derivative of the S protein RBD sequence comprises anamino acid substitution N501 T. In particular embodiments, the encodedfunctional derivative of the S protein RBD sequence comprises an aminoacid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ IDNO:26. In particular embodiments, the therapeutic nucleic acid encodinga functional derivative of the RBD sequence of the S protein ofcoronavirus SARS-CoV-2 comprises a DNA coding sequence of SEQ ID NO:27.In particular embodiments, the therapeutic nucleic acid encoding afunctional derivative of the RBD sequence of the S protein ofcoronavirus SARS-CoV-2 comprises a RNA sequence transcribed from the DNAcoding sequence of SEQ D NO:27. In particular embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, thetherapeutic nucleic acid is an mRNA molecule.

Without being bound by the theory, it is contemplated that in thecoronavirus spike structure, three 51 heads sit on top of a trimeric S2stalk. Between the two major 51 domains, S1-CTD is located at the verytop of the spike, whereas S1-NTD directly contacts and structurallyconstrains S2. Accordingly, in some embodiments, the therapeutic nucleicacid of the present disclosure encodes a functional derivative of the Sprotein. In some embodiments, the therapeutic nucleic acid encodes afusion protein comprising an S protein or a fragment thereof fused to atrimmerization peptide, such that the fusion protein is capable offorming a trimeric complex comprising three copies of the S protein orfragment thereof. In some embodiments, the therapeutic nucleic acidencodes a fusion protein comprising an ectodomain of the S protein fusedto a trimmerization peptide, wherein the fusion protein is capable offorming a trimeric complex comprising three copies of the ectodomain. Insome embodiments, the therapeutic nucleic acid encodes a fusion proteincomprising a RBD of the S protein fused to a trimmerization peptide,wherein the fusion protein is capable of forming a trimeric complexcomprising three copies of the RBD. In some embodiments, the therapeuticnucleic acid encodes a fusion protein comprising a S1-CTD fused to atrimmerization peptide wherein the fusion protein is capable of forminga trimeric complex comprising three copies of the S1-CTD. In someembodiments, the S protein or fragment thereof is fused to atrimmerization peptide via a peptidic linker. Table 3 shows exemplarytrimmerization peptide and linker peptide that can be used in connectionwith the present disclosure, and sequences of fusion proteins.

TABLE 3 Exemplary sequences of linker peptides,trimmerization peptides, and SARS-CoV- 2 antigens. SEQUENCE NAMEAMINO ACID OR NUCLEIC (SEQ ID NO:) ACID SEQUENCE (G3S)2 linker GGGSGGGSpeptide amino acid sequence (SEQ ID NO: 28) (G35)2 linkerGGAGGAGGAAGTGGAGGAGGAAGT peptide coding sequence (SEQ ID NO: 29)Trimmerization GYIPEAPRDGQAYVRKDGEWVLLS peptide TFLG amino acid sequence(SEQ ID NO: 30) Trimmerization GGCTATATTCCGGAAGCGCCGCGC peptideGATGGCCAGGCGTATGTGCGCAAA coding GATGGCGAATGGGTGCTGCTGAGC sequenceACCTTTCTGGGC (SEQ ID NO: 31) SARS-CoV-2 QCVNLTTRTQLPPAYTNSFTRGVY spikeYPDKVFRSSVLHSTQDLFLPFFSN protein ECD VTWFHAIHVSGTNGTKRFDNPVLPwith C terminal FNDGVYFASTEKSMIRGWIFGTTL fusion ofDSKTQSLLIVNNATNVVIKVCEFQ a (G3S)2 FCNDPFLGVYYHKNNKSWMESEFR linker and aVYSSANNCTFEYVSQPFLMDLEGK trimmerization QGNFKNLREFVFKNIDGYFKIYSK peptideHTPINLVRDLPQGFSALEPLVDLP amino acid IGINITRFQTLLALHRSYLTPGDS sequenceSSGWTAGAAAYYVGYLQPRTFLLK (SEQ ID YNENGTITDAVDCALDPLSETKCT NO: 32)LKSFTVEKGIYQTSNFRVQPTESI VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASF STFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYN YKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL HAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFG RDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQD VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYEC DIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNN SIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGS FCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQIL PDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQK FNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQM AYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQD VVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLIT GRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKG YHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREG VFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDP LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNE VAKNLNESLIDLQELGKYEQYIKG GGSGGGSGYIPEAPRDGQAYVRKD GEWVLLSTFLG SARS-CoV-2 CAGTGTGTTAATCTTACAACCAGAspike protein ACTCAATTACCCCCTGCATACACT ECD with CAATTCTTTCACACGTGGTGTTTAT terminal TACCCTGACAAAGTTTTCAGATCC fusion ofTCAGTTTTACATTCAACTCAGGAC a (G3S)2 TTGTTCTTACCTTTCTTTTCCAAT linker and aGTTACTTGGTTCCATGCTATACAT trimmerization GTCTCTGGGACCAATGGTACTAAGpeptide coding AGGTTTGATAACCCTGTCCTACCA sequenceTTTAATGATGGTGTTTATTTTGCT (SEQ ID TCCACTGAGAAGTCTAACATAATA NO: 33)AGAGGCTGGATTTTTGGTACTACT TTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAAT GTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTG GGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTC AGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAG CCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTT AGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCT AAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCG GCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGG TTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGAT TCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTAT CTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATT ACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGT ACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAAC TTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACA AACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCT GTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTAT TCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGA GTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCA GATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGG CAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTT ACAGGCTGCGTTATAGCTTGGAACTCTAACAATCTTGATTCTAAGGTT GGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTC AAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGC ACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAA TCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGA GTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGT GGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTC AACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAA AAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACT GATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCA TGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCT AACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCT GTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCT ACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCT GAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGT ATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGT AGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCA GAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAAT TTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAG ACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGC AGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGT GCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTT TTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTT GGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGC AAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCA GATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCT GCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTG CCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTG TTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCA TTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATT GGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAAC CAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACA GCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAA GCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATT TCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCT GAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAG ACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCT GCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCA AAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCT CAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCT GCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGA AAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACAC TGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACA GACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTC AACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAG GAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGAT TTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAA GAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTC ATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAAGGAGGA GGAAGTGGAGGAGGAAGTGGCTATATTCCGGAAGCGCCGCGCGATGGC CAGGCGTATGTGCGCAAAGATGGCGAATGGGTGCTGCTGAGCACCTTT CTGGGC SARS-CoV-2 NITNLCPFGEVFNATRFASVYAWNspike protein RKRISNCVADYSVLYNSASFSTFK RBD-2 with C-CYGVSPTKLNDLCFTNVYADSFVI terminal RGDEVRQIAPGQTGKIADYNYKLP fusion ofDDFTGCVIAWNSNNLDSKVGGNYN a (G3S)2 YLYRLFRKSNLKPFERDISYEIYQ linkerAGSTPCNGVEGFNCYFPLQSYGFQ and a PTNGVGYQPYRVVVLSFELLHAPA trimmerizationTVCGPKKGGGSGGGSG YIPEAPRD peptide GQAYVRKDGEWVLLSTFLG amino acidsequence (SEQ ID NO: 34) SARS-CoV-2 AATATTACAAACTTGTGCCCTTTTspike protein GGTGAAGTTTTTAACGCCACCAGA RBD-2 with C-TTTGCATCTGTTTATGCTTGGAAC terminal AGGAAGAGAATCAGCAACTGTGTT fusion ofGCTGATTATTCTGTCCTATATAAT a (G3S)2 TCCGCATCATTTTCCACTTTTAAG linkerTGTTATGGAGTGTCTCCTACTAAA and a TTAAATGATCTCTGCTTTACTAAT trimmerizationGTCTATGCAGATTCATTTGTAATT peptide AGAGGTGATGAAGTCAGACAAATC codingGCTCCAGGGCAAACTGGAAAGATT sequence GCTGATTATAATTATAAATTACCA (SEQ IDGATGATTTTACAGGCTGCGTTATA NO: 35) GCTTGGAACTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAAT TACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGA GATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGT GTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAA CCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCT TTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGGGA GGAGGAAGTGGAGGAGGAAGTGGCTATATTCCGGAAGCGCCGCGCGAT GGCCAGGCGTATGTGCGCAAAGATGGCGAATGGGTGCTGCTGAGCACC TTTCTGGGC

In some embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising the S protein of the coronavirus SARS-CoV-2 or afunctional derivative thereof fused to a trimmerization peptide. In someembodiments, the fusion between the S protein and the trimmerizationpeptide is via a peptide linker. In specific embodiments, the S proteinor functional derivative thereof comprises the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:20. In specific embodiments, the peptide linkercomprises the amino acid sequence of SEQ ID NO:28. In some embodiments,the trimmerization peptide comprises the amino acid sequence of SEQ IDNO:30.

In some embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising the ectodomain (ECD) of S protein of the coronavirusSARS-CoV-2 or a functional derivative thereof fused to a trimmerizationpeptide. In some embodiments, the fusion between the ectodomain of the Sprotein and the trimmerization peptide is via a peptide linker. Inspecific embodiments, the ectodomain of S protein or functionalderivative thereof comprises the amino acid sequence of SEQ ID NO:4 orSEQ ID NO:21. In specific embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO:28. In some embodiments, thetrimmerization peptide comprises the amino acid sequence of SEQ IDNO:30.

In some embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising an ectodomain of the S protein of the coronavirusSARS-CoV-2 or a functional derivative thereof fused to a trimmerizationpeptide. In particular embodiments, the fusion protein has an amino acidsequence of SEQ ID NO:32. In particular embodiments, the therapeuticnucleic acid encodes a fusion protein comprising an ectodomain of the Sprotein of the SARS-CoV-2 fused to a trimmerization peptide, wherein thenucleic acid comprises a DNA coding sequence of SEQ ID NO:33. Inparticular embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising an ectodomain of the S protein of the SARS-CoV-2fused to a trimmerization peptide, wherein the nucleic acid comprises aRNA sequence transcribed from the DNA coding sequence of SEQ ID NO:33.In some embodiments, the RNA sequence is in vitro transcribed. Inparticular embodiments, the nucleic acid molecule is an mRNA molecule.

In some embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising the S1 subunit of the S protein of the coronavirusSARS-CoV-2 or a functional derivative thereof fused to a trimmerizationpeptide. In some embodiments, the fusion between the ectodomain of the Sprotein and the trimmerization peptide is via a peptide linker. Inspecific embodiments, the S1 subunit of S protein or functionalderivative thereof comprises the amino acid sequence of SEQ ID NO:6 orSEQ ID NO:22. In specific embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO:28. In some embodiments, thetrimmerization peptide comprises the amino acid sequence of SEQ IDNO:30.

In some embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising a receptor binding domain (RBD) sequence of the Sprotein of the coronavirus SARS-CoV-2 or a functional derivative thereoffused to a trimmerization peptide. In some embodiments, the fusionbetween the RBD sequence of the S protein and the trimmerization peptideis via a peptide linker. In specific embodiments, the RBD sequence of Sprotein or functional derivative thereof comprises the amino acidsequence selected from SEQ ID NOS:8, 10, 12, 14, 23, 24, 25 and 26. Inspecific embodiments, the peptide linker comprises the amino acidsequence of SEQ ID NO:28. In some embodiments, the trimmerizationpeptide comprises the amino acid sequence of SEQ ID NO:30.

In particular embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising a RBD sequence of the S protein of the SARS-CoV-2fused to a trimmerization peptide, wherein the fusion protein has anamino acid sequence of SEQ ID NO:34. In particular embodiments, thetherapeutic nucleic acid encodes a fusion protein comprising the RBD ofthe S protein of the SARS-CoV-2 fused to a trimmerization peptide,wherein the nucleic acid comprises a DNA coding sequence of SEQ IDNO:35. In particular embodiments, the therapeutic nucleic acid encodes afusion protein comprising the RBD of the S protein of the SARS-CoV-2fused to a trimmerization peptide, wherein the nucleic acid comprises aRNA sequence transcribed from the DNA coding sequence of SEQ ID NO:35.In some embodiments, the RNA sequence is in vitro transcribed. Inparticular embodiments, the nucleic acid molecule is an mRNA molecule.

In some embodiments, the therapeutic nucleic acid encodes a fusionprotein comprising a receptor binding motif (RBM) sequence of the Sprotein of the coronavirus SARS-CoV-2 or a functional derivative thereoffused to a trimmerization peptide. In some embodiments, the fusionbetween the RBM sequence of the S protein and the trimmerization peptideis via a peptide linker. In specific embodiments, the RBM sequence of Sprotein or functional derivative thereof comprises the amino acidsequence of SEQ ID NO:16. In specific embodiments, the peptide linkercomprises the amino acid sequence of SEQ ID NO:28. In some embodiments,the trimmerization peptide comprises the amino acid sequence of SEQ IDNO:30.

Without being bound by the theory, it is contemplated that the N proteinof coronavirus comprises an N-terminal domain (N-NTD) and a C-terminaldomain (N-CTD), which are interspersed by several intrinsicallydisordered regions (IDRs). For example, SARS-CoV N protein has threeIDRs at residues 1-44, 182-247, and 366-422, respectively, and an N-NTDlocated at residues 45-181, and an N-CTD located at residues 248-365.

Accordingly, in some embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the coronavirus N protein, or an immunogenicfragment of the N protein, or a functional derivative of the N proteinor the immunogenic fragment thereof. In specific embodiments, thetherapeutic nucleic acid encodes the full-length N protein. In specificembodiments, the therapeutic nucleic acid encodes one or moreimmunogenic fragments of the N protein selected from the N-NTD, N-CTDand IDRs.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the nucleocapsid (N) protein of coronavirusSARS-CoV-2, and wherein the N protein has an amino acid sequence of SEQID NO:18. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the N protein of coronavirus SARS-CoV-2, andwherein the therapeutic nucleic acid comprises a DNA coding sequence ofSEQ ID NO:19. In particular embodiments, the therapeutic nucleic acid ofthe present disclosure encodes the N protein of coronavirus SARS-CoV-2,and wherein the therapeutic nucleic acid comprises a RNA sequencetranscribed from the DNA coding sequence of SEQ ID NO:19. In someembodiments, the RNA sequence is in vitro transcribed. In particularembodiments, the nucleic acid molecule is an mRNA molecule.

Without being bound by the theory, it is contemplated that a fusionprotein comprising a viral peptide or polypeptide fused to animmunoglobulin Fc region can enhance immunogenicity of the viral peptideor polypeptide. Accordingly, in some embodiments, the therapeuticnucleic acid molecule of the present disclosure encodes a fusion proteincomprising a viral peptide or protein derived from a coronavirus fusedwith an Fc region of an immunoglobulin. In particular embodiments, theviral peptide or protein is one or more selected from (a) the N protein,(b) the M protein, (c) the E protein, (d) the S protein, (e) the HEprotein, (f) an immunogenic fragment of any one of (a) to (e), and (g) afunctional derivative of any one of (a) to (f). In particularembodiments, the immunoglobulin is human immunoglobulin (Ig). Inparticular embodiments, the immunoglobulin is human IgG, IgA, IgD, IgE,or IgM. In particular embodiments, the immunoglobulin is human IgG1,IgG2, IgG3 or IgG4. In some embodiments, the immunoglobulin Fc is fusedto the N terminus of the viral peptide or polypeptide. In otherembodiments, the immunoglobulin Fc is fused to the C terminus of theviral peptide or polypeptide.

Without being bound by theory, it is contemplated that a signal peptidecan mediate transportation of a polypeptide fused thereto to particularlocations of a cell. Accordingly, in some embodiments, the therapeuticnucleic acid molecule of the present disclosure encodes a fusion proteincomprising a viral peptide or protein fused to a signal peptide. Inparticular embodiments, the viral peptide or protein is one or moreselected from (a) the N protein, (b) the M protein, (c) the E protein,(d) the S protein, (e) the HE protein, (f) an immunogenic fragment ofany one of (a) to (e), and (g) a functional derivative of any one of (a)to (f). In some embodiments, the signal peptide is fused to the Nterminus of the viral peptide or polypeptide. In other embodiments, thesignal peptide is fused to the C terminus of the viral peptide orpolypeptide. Table 4 shows exemplary sequences for signal peptides thatcan be use in connection with the present disclosure, and exemplarySARS-CoV-2 antigenic sequences comprising the signal peptides.

TABLE 4 Exemplary sequences of signal peptides and SARS-CoV-2 antigens.SEQUENCE NAME (SEQ ID AMINO ACID OR NUCLEIC NO:) ACID SEQUENCESARS-CoV-2 MFVFLVLLPLVSS spike protein native signal peptide amino acidsequence (SEQ ID NO: 36) SARS-CoV-2 ATGTTTGTTTTTCTTGTTTTATTGspike protein CCATTAGTCTCTAGT native signal peptide codingsequence (SEQ ID NO: 37) Human IgE signal MDWTWILFLVAAATRVHSpeptide amino acid sequence (SEQ ID NO: 38) Human IgE signalATGGACTGGACCTGGATTCTCTTC peptide coding TTGGTGGCAGCAGCCACGCGAGTCsequence (SEQ ID CACTCC NO: 39) SARS-CoV-2 QCVNLTTRTQLPPAYTNSFTRGVYspike protein YPDKVFRSSVLHSTQDLFLPFFSN without nativeVTWFHAIHVSGTNGTKRFDNPVLP signal peptide FNDGVYFASTEKSNIIRGWIFGTTamino acid LDSKTQSLLIVNNATNVVIKVCEF sequence (SEQ IDQFCNDPFLGVYYHKNNKSWMESE NO: 40) FRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTP GDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRF ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV YADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFP LQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDI TPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRV YSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRR ARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSM TKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQ EVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVT LADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTS ALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLI ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFG AISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIR ASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVT YVPAQEKNFTTAPAICEIDGKAHF PREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNT VYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEID RLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTI MLCCMTSCCSCLKGCCSCGSCCKF DEDDSEPVLKGVKLHYTSARS-CoV-2 ATGTTTGTTTTTCTTGTTTTATTG spike proteinCCATTAGTCTCTAGTCAGTGTGTT without native AATCTTACAACCAGAACTCAATTAsignal peptide CCCCCTGCATACACTAATTCTTTC coding sequenceACACGTGGTGTTTATTACCCTGAC (SEQ ID NO: 41) AAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTA CCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGG ACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGAT GGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGG ATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTT AATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGT AATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGG ATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTT GAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGT AATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTAT TTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTC CCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGT ATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTAT TTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCT TATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAAT GAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTC TCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATC TATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGA TTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCC ACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAAC TGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACT TTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTT ACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGA CAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAA TTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAACTCTAACAAT CTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTT AGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATC TATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGT TACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGT TACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCA CCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAAC AAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTT ACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGAC ATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATT CTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCA GGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAAC TGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACT TGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGC TGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATA CCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCT CCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACT ATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATT GCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCA GTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGT GATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGT ACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAA AACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCA CCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGAT CCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAAC AAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGC CTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAAC GGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAA TACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTT GGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTAT AGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAA AAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGAC TCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTC AACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCC AATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTT GACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGA CTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCA GAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGT GTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCAT CTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCAT GTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCC ATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTT TCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCA CAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTT GTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAA TTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACA TCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTT GTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAAT TTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAG TATATAAAA SARS-CoV-2 MFVFLVLLPLVSSQCVNLTTRTQLPspike protein PAYTNSFTRGVYYPDKVFRSSVLH ectodomain (ECD)STQDLFLPFFSNVTWFHAIHVSGT with native signal NGTKRFDNPVLPFNDGVYFASTEKpeptide amino acid SNIIRGWIFGTTLDSKTQSLLIVN sequence (SEQ IDNATNWIKVCEFQFCNDPFLGVYYH NO: 42) KNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVF KNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL ALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVD CALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPF GEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTK LNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG VEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKST NLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDP QTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHAD QLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIA VEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIE DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTD EMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNV LYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVK QLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQL IRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGW FLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNF YEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFK NHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKY EQYIK SARS-CoV-2 ATGTTTGTTTTTCTTGTTTTATTGspike protein CCATTAGTCTCTAGTCAGTGTGTT ectodomain (ECD)AATCTTACAACCAGAACTCAATTA with native signal CCCCCTGCATACACTAATTCTTTCpeptide coding ACACGTGGTGTTTATTACCCTGAC sequence (SEQ IDAAAGTTTTCAGATCCTCAGTTTTA NO: 43) CATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGG TTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGAT AACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAG AAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCG AAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATT AAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTAT TACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTAT TCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTT ATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTT GTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACG CCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAA CCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACT TTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA GGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCT AGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCT GTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAA TCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTC CAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGC CCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCT TGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTA TATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCT ACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTT GTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGA AAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGC GTTATAGCTTGGAACTCTAACAATCTTGATTCTAAGGTTGGTGGTAAT TATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTT GAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGT AATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGT TTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTA CTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAA AAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAAT GGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTG CCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTC CGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTT GGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTT GCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATT CATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCT AATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTC AACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCT AGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCT AGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCA GTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATT AGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTA GATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTT TTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACT GGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAA GTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTT AATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCA TTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGC TTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGAC CTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTG CTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGT ACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATA CCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACA CAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAAT AGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCA CTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAAC ACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTT TTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAA ATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTG ACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTT GCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTT GATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCA CCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAA AAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACAC TTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTA ACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACA TTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACA GTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTA GATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGAC ATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGAC CGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTC CAAGAACTTGGAAAGTATGAGCAG TATATAAAA SARS-CoV-2MFVFLVLLPLVSSQCVNLTTRTQL spike protein S1 PPAYTNSFTRGVYYPDKVFRSSVLsubunit with native HSTQDLFLPFFSNVTWFHAIHVSG signal peptideTNGTKRFDNPVLPFNDGVYFASIE amino acid KSNIIRGWIFGTTLDSKTQSLLIVsequence (SEQ ID NNATNVVIKVCEFQFCNDPFLGVY NO: 44)YHKNNKSWMESEFRVYSSANNCTF EYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDL PQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAA YYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPIESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR QIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVL TESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG CLIGAEHVNNSYECDIPIGAGICA SYQTQTNSPRRARSARS-CoV-2 ATGTTTGTTTTTCTTGTTTTATTG spike protein S1CCATTAGTCTCTAGTCAGTGTGTT subunit with native AATCTTACAACCAGAACTCAATTAsignal peptide CCCCCTGCATACACTAATTCTTTC coding sequenceACACGTGGTGTTTATTACCCTGAC (SEQ ID NO: 45) AAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTA CCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGG ACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGAT GGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGG ATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTT AATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGT AATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGG ATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTT GAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGT AATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTAT TTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTC CCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGT ATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTAT TTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCT TATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAAT GAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTC TCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATC TATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGA TTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCC ACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAAC TGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACT TTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTT ACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGA CAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAA TTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAAT CTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTT AGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATC TATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGT TACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGT TACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCA CCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAAC AAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTT ACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGAC ATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATT CTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCA GGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAAC TGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACT TGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGC TGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATA CCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCT CCTCGGCGGGCACGT

In particular embodiments, the signal peptide is encoded by a gene ofthe coronavirus from which the viral peptide or polypeptide is derived.In particular embodiments, a signal peptide encoded by a gene of thecoronavirus is fused to a viral peptide or polypeptide encoded by adifferent gene of the coronavirus. In other embodiments, a signalpeptide encoded by a gene of the coronavirus is fused to a viral peptideor polypeptide encoded by the same gene of the coronavirus. For example,in some embodiments, a signal peptide having amino acid sequence ofMFVFLVLLPLVSS (SEQ ID NO:36) is fused to the viral peptide orpolypeptide encoded by the nucleic acid molecule of the presentdisclosure. In various embodiments, the viral peptide or protein is oneor more selected from (a) the N protein, (b) the M protein, (c) the Eprotein, (d) the S protein, (e) the HE protein, (f) an immunogenicfragment of any one of (a) to (e), and (g) a functional derivative ofany one of (a) to (f).

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the S protein of coronavirus SARS-CoV-2 without thenative signal peptide. In particular embodiments, the encoded S proteincomprises an amino acid sequence of SEQ ID NO:40. In particularembodiments, the therapeutic nucleic acid of the present disclosureencodes the S protein of coronavirus SARS-CoV-2 having a signal peptide,and wherein the therapeutic nucleic acid comprises a DNA coding sequenceof SEQ ID NO:41. In particular embodiments, the therapeutic nucleic acidof the present disclosure encodes the S protein of coronavirusSARS-CoV-2 having a signal peptide, and wherein the therapeutic nucleicacid comprises a RNA sequence transcribed from the DNA coding sequenceof SEQ ID NO:41. In some embodiments, the RNA sequence is in vitrotranscribed. In particular embodiments, the nucleic acid molecule is anmRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the ectodomain (ECD) of the S protein of coronavirusSARS-CoV-2 having a signal peptide. In particular embodiments, theencoded ectodomain of S protein comprises an amino acid sequence of SEQID NO:42. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the ectodomain of the S protein ofcoronavirus SARS-CoV-2 having a signal peptide, and wherein thetherapeutic nucleic acid comprises a DNA coding sequence of SEQ IDNO:43. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the ectodomain of the S protein ofcoronavirus SARS-CoV-2 having a signal peptide, and wherein thetherapeutic nucleic acid comprises a RNA sequence transcribed from theDNA coding sequence of SEQ ID NO:43. In some embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, the nucleicacid molecule is an mRNA molecule.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure encodes the S1 subunit of the S protein of coronavirusSARS-CoV-2 having a signal peptide. In particular embodiments, theencoded S1 subunit of S protein comprises an amino acid sequence of SEQID NO:44. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the S1 subunit of the S protein ofcoronavirus SARS-CoV-2 having a signal peptide, and wherein thetherapeutic nucleic acid comprises a DNA coding sequence of SEQ IDNO:45. In particular embodiments, the therapeutic nucleic acid of thepresent disclosure encodes the S1 subunit of the S protein ofcoronavirus SARS-CoV-2 having a signal peptide, and wherein thetherapeutic nucleic acid comprises a RNA sequence transcribed from theDNA coding sequence of SEQ ID NO:45. In some embodiments, the RNAsequence is in vitro transcribed. In particular embodiments, the nucleicacid molecule is an mRNA molecule.

In other embodiments, the signal peptide is encoded by an exogenous genesequence that does not present in the coronavirus from which the viralpeptide or polypeptide is derived. In some embodiments, a heterologoussignal peptide replaces a homologous signal peptide in the fusionprotein encoded by the nucleic acid molecule of the present disclosure.In specific embodiments, the signal peptide is encoded by a mammaliangene. In specific embodiments, the signal peptide is encoded by humanImmunoglobulin gene. In specific embodiments, the signal peptide isencoded by human IgE gene. For example, in some embodiments, a signalpeptide having amino acid sequence of MDWTWILFLVAAATRVHS (SEQ ID NO:38)is fused to the viral peptide or polypeptide encoded by the nucleic acidmolecule of the present disclosure. In various embodiments, the viralpeptide or protein is one or more selected from (a) the N protein, (b)the M protein, (c) the E protein, (d) the S protein, (e) the HE protein,(f) an immunogenic fragment of any one of (a) to (e), and (g) afunctional derivative of any one of (a) to (f).

6.3.2 5′-Cap Structure

Without being bound by the theory, it is contemplated that, a 5′-capstructure of a polynucleotide is involved in nuclear export andincreasing polynucleotide stability and binds the mRNA Cap BindingProtein (CBP), which is responsible for polynucleotide stability in thecell and translation competency through the association of CBP withpoly-A binding protein to form the mature cyclic mRNA species. The5′-cap structure further assists the removal of 5′-proximal intronsremoval during mRNA splicing. Accordingly, in some embodiments, thenucleic acid molecules of the present disclosure comprise a 5′-capstructure.

Nucleic acid molecules may be 5′-end capped by the endogenoustranscription machinery of a cell to generate a 5′-ppp-5′-triphosphatelinkage between a terminal guanosine cap residue and the 5′-terminaltranscribed sense nucleotide of the polynucleotide. This 5′-guanylatecap may then be methylated to generate an N7-methyl-guanylate residue.The ribose sugars of the terminal and/or anteterminal transcribednucleotides of the 5′ end of the polynucleotide may optionally also be2′-O-methylated. 5′-decapping through hydrolysis and cleavage of theguanylate cap structure may target a nucleic acid molecule, such as anmRNA molecule, for degradation.

In some embodiments, the nucleic acid molecules of the presentdisclosure comprise one or more alterations to the natural 5′-capstructure generated by the endogenous process. Without being bound bythe theory, a modification on the 5′-cap may increase the stability ofpolynucleotide, increase the half-life of the polynucleotide, and couldincrease the polynucleotide translational efficiency.

Exemplary alterations to the natural 5′-Cap structure include generationof a non-hydrolyzable cap structure preventing decapping and thusincreasing polynucleotide half-life. In some embodiments, because capstructure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiesterlinkages, in some embodiments, modified nucleotides may be used duringthe capping reaction. For example, in some embodiments, a VacciniaCapping Enzyme from New England Biolabs (Ipswich, Mass.) may be usedwith α-thio-guanosine nucleotides according to the manufacturer'sinstructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.Additional modified guanosine nucleotides may be used, such asα-methyl-phosphonate and seleno-phosphate nucleotides.

Additional exemplary alterations to the natural 5′-Cap structure alsoinclude modification at the 2′- and/or 3′-position of a capped guanosinetriphosphate (GTP), a replacement of the sugar ring oxygen (thatproduced the carbocyclic ring) with a methylene moiety (CH₂), amodification at the triphosphate bridge moiety of the cap structure, ora modification at the nucleobase (G) moiety.

Additional exemplary alterations to the natural 5′-cap structureinclude, but are not limited to, 2′-O-methylation of the ribose sugarsof 5′-terminal and/or 5′-anteterminal nucleotides of the polynucleotide(as mentioned above) on the 2′-hydroxy group of the sugar. Multipledistinct 5′-cap structures can be used to generate the 5′-cap of apolynucleotide, such as an mRNA molecule. Additional exemplary 5′-Capstructures that can be used in connection with the present disclosurefurther include those described in International Patent Publication Nos.WO2008127688, WO 2008016473, and WO 2011015347, the entire contents ofeach of which are incorporated herein by reference.

In various embodiments, 5′-terminal caps can include cap analogs. Capanalogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e., endogenous, wild-type, orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e., non-enzymatically) orenzymatically synthesized and/linked to a polynucleotide.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanosines linked by a 5′-5′-triphosphate group, wherein one guanosinecontains an N7-methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷G-3′mppp-G,which may equivalently be designated 3′ 0-Me-m7G(5′)ppp(5′)G). The 3′-Oatom of the other, unaltered, guanosine becomes linked to the5′-terminal nucleotide of the capped polynucleotide (e.g., an mRNA). TheN7- and 3′-O-methlyated guanosine provides the terminal moiety of thecapped polynucleotide (e.g., mRNA). Another exemplary cap structure ismCAP, which is similar to ARCA but has a 2′-O-methyl group on guanosine(i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine,m⁷Gm-ppp-G).

In some embodiments, a cap analog can be a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog may be modified atdifferent phosphate positions with a boranophosphate group or aphophoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110, the entire content of which is hereinincorporated by reference in its entirety.

In some embodiments, a cap analog can be a N7-(4-chlorophenoxyethyl)substituted dinucleotide cap analog known in the art and/or describedherein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituteddinucleotide cap analogs include aN7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m3′-OG(5′)ppp(5′)G cap analog (see, e.g., thevarious cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the entire content of which is herein incorporated byreference). In other embodiments, a cap analog useful in connection withthe nucleic acid molecules of the present disclosure is a4-chloro/bromophenoxyethyl analog.

In various embodiments, a cap analog can include a guanosine analog.Useful guanosine analogs include but are not limited to inosine,N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine.

Without being bound by the theory, it is contemplated that while capanalogs allow for the concomitant capping of a polynucleotide in an invitro transcription reaction, up to 20% of transcripts remain uncapped.This, as well as the structural differences of a cap analog from thenatural 5′-cap structures of polynucleotides produced by the endogenoustranscription machinery of a cell, may lead to reduced translationalcompetency and reduced cellular stability.

Accordingly, in some embodiments, a nucleic acid molecule of the presentdisclosure can also be capped post-transcriptionally, using enzymes, inorder to generate more authentic 5′-cap structures. As used herein, thephrase “more authentic” refers to a feature that closely mirrors ormimics, either structurally or functionally, an endogenous or wild typefeature. That is, a “more authentic” feature is better representative ofan endogenous, wild-type, natural or physiological cellular function,and/or structure as compared to synthetic features or analogs of theprior art, or which outperforms the corresponding endogenous, wild-type,natural, or physiological feature in one or more respects. Non-limitingexamples of more authentic 5′-cap structures useful in connection withthe nucleic acid molecules of the present disclosure are those which,among other things, have enhanced binding of cap binding proteins,increased half-life, reduced susceptibility to 5′-endonucleases, and/orreduced 5′-decapping, as compared to synthetic 5′-cap structures knownin the art (or to a wild-type, natural or physiological 5′-capstructure). For example, in some embodiments, recombinant Vaccinia VirusCapping Enzyme and recombinant 2′-O-methyltransferase enzyme can createa canonical 5′-5′-triphosphate linkage between the 5′-terminalnucleotide of a polynucleotide and a guanosine cap nucleotide whereinthe cap guanosine contains an N7-methylation and the 5′-terminalnucleotide of the polynucleotide contains a 2′-O-methyl. Such astructure is termed the Cap1 structure. This cap results in a highertranslational-competency, cellular stability, and a reduced activationof cellular pro-inflammatory cytokines, as compared, e.g., to other5′cap analog structures known in the art. Other exemplary cap structuresinclude 7mG(5′)ppp(5′)N,pN2p (Cap 0), 7mG(5′)ppp(5′)NlmpNp (Cap 1),7mG(5′)-ppp(5′)NlmpN2mp (Cap 2), andm(7)Gpppm(3)(6,6,2′)Apm(2′)Apm(2′)Cpm(2)(3,2′)Up (Cap 4).

Without being bound by the theory, it is contemplated that the nucleicacid molecules of the present disclosure can be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the nucleic acid molecules may be capped.

6.3.3 Untranslated Regions (UTRs)

In some embodiments, the nucleic acid molecules of the presentdisclosure comprise one or more untranslated regions (UTRs). In someembodiments, an UTR is positioned upstream to a coding region in thenucleic acid molecule, and is termed 5′-UTR. In some embodiments, an UTRis positioned downstream to a coding region in the nucleic acidmolecule, and is termed 3′-UTR. The sequence of an UTR can be homologousor heterologous to the sequence of the coding region found in a nucleicacid molecule. Multiple UTRs can be included in a nucleic acid moleculeand can be of the same or different sequences, and/or genetic origin.According to the present disclosure, any portion of UTRs in a nucleicacid molecule (including none) can be codon optimized and any mayindependently contain one or more different structural or chemicalmodification, before and/or after codon optimization.

In some embodiments, a nucleic acid molecule of the present disclosure(e.g., mRNA) comprises UTRs and coding regions that are homologous withrespect to each other. In other embodiments, a nucleic acid molecule ofthe present disclosure (e.g., mRNA) comprises UTRs and coding regionsthat are heterologous with respect to each other. In some embodiments,to monitor the activity of a UTR sequence, a nucleic acid moleculecomprising the UTR and a coding sequence of a detectable probe can beadministered in vitro (e.g., cell or tissue culture) or in vivo (e.g.,to a subject), and an effect of the UTR sequence (e.g., modulation onthe expression level, cellular localization of the encoded product, orhalf-life of the encoded product) can be measured using methods known inthe art.

In some embodiments, the UTR of a nucleic acid molecule of the presentdisclosure (e.g., mRNA) comprises at least one translation enhancerelement (TEE) that functions to increase the amount of polypeptide orprotein produced from the nucleic acid molecule. In some embodiments,the TEE is located in the 5′-UTR of the nucleic acid molecule. In otherembodiments, the TEE is located at the 3′-UTR of the nucleic acidmolecule. In yet other embodiments, at least two TEE are located at the5′-UTR and 3′-UTR of the nucleic acid molecule respectively. In someembodiments, a nucleic acid molecule of the present disclosure (e.g.,mRNA) can comprise one or more copies of a ‘l’EE sequence or comprisemore than one different TEE sequences. In some embodiments, differentTEE sequences that are present in a nucleic acid molecule of the presentdisclosure can be homologues or heterologous with respect to oneanother.

Various TEE sequences that are known in the art and can be used inconnection with the present disclosure. For example, in someembodiments, the TEE can be an internal ribosome entry site (IRES),HCV-IRES or an IRES element. Chappell et al. Proc. Natl. Acad. Sci. USA101:9590-9594, 2004; Zhou et al. Proc. Natl. Acad. Sci. 102:6273-6278,2005. Additional internal ribosome entry site (IRES) that can be used inconnection with the present disclosure include but are not limited tothose described in U.S. Pat. No. 7,468,275, U.S. Patent Publication No.2007/0048776 and U.S. Patent Publication No. 2011/0124100 andInternational Patent Publication No. WO2007/025008 and InternationalPatent Publication No. WO2001/055369, the content of each of which isenclosed herein by reference in its entirety. In some embodiments, theTEE can be those described in Supplemental Table 1 and in SupplementalTable 2 of Wellensiek et al Genome-wide profiling of humancap-independent translation-enhancing elements, Nature Methods, 2013August; 10(8): 747-750; the content of which is incorporated byreference in its entirety.

Additional exemplary TEEs that can be used in connection with thepresent disclosure include but are not limited to the TEE sequencesdisclosed in U.S. Pat. Nos. 6,310,197, 6,849,405, U.S. Pat. Nos.7,456,273, 7,183,395, U.S. Patent Publication No. 2009/0226470, U.S.Patent Publication No. 2013/0177581, U.S. Patent Publication No.2007/0048776, U.S. Patent Publication No. 2011/0124100, U.S. PatentPublication No. 2009/0093049, International Patent Publication No.WO2009/075886, International Patent Publication No. WO2012/009644, andInternational Patent Publication No. WO1999/024595, International PatentPublication No. WO2007/025008, International Patent Publication No.WO2001/055371, European Patent No. 2610341, European Patent No. 2610340,the content of each of which is enclosed herein by reference in itsentirety.

In various embodiments, a nucleic acid molecule of the presentdisclosure (e.g., mRNA) comprises at least one UTR that comprises atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18 at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55 or more than 60 TEE sequences. Insome embodiments, the TEE sequences in the UTR of a nucleic acidmolecule are copies of the same TEE sequence. In other embodiments, atleast two TEE sequences in the UTR of a nucleic acid molecule are ofdifferent TEE sequences. In some embodiments, multiple different TEEsequences are arranged in one or more repeating patterns in the UTRregion of a nucleic acid molecule. For illustrating purpose only, arepeating pattern can be, for example, ABABAB, AABBAABBAABB, ABCABCABC,or the like, where in these exemplary patterns, each capitalized letter(A, B, or C) represents a different TEE sequence. In some embodiments,at least two TEE sequences are consecutive with one another (i.e., nospacer sequence in between) in a UTR of a nucleic acid molecule. Inother embodiments, at least two TEE sequences are separated by a spacersequence. In some embodiments, a UTR can comprise a TEE sequence-spacersequence module that is repeated at least once, at least twice, at least3 times, at least 4 times, at least 5 times, at least 6 times, at least7 times, at least 8 times, at least 9 times, or more than 9 times in theUTR. In any of the embodiments described in this paragraph, the UTR canbe a 5′-UTR, a 3′-UTR or both 5′-UTR and 3′-UTR of a nucleic acidmolecule.

In some embodiments, the UTR of a nucleic acid molecule of the presentdisclosure (e.g., mRNA) comprises at least one translation suppressingelement that functions to decrease the amount of polypeptide or proteinproduced from the nucleic acid molecule. In some embodiments, the UTR ofthe nucleic acid molecule comprises one or more miR sequences orfragment thereof (e.g., miR seed sequences) that are recognized by oneor more microRNA. In some embodiments, the UTR of the nucleic acidmolecule comprises one or more stem-loop structure that downregulatestranslational activity of the nucleic acid molecule. Other mechanismsfor suppressing translational activities associated with a nucleic acidmolecules are known in the art. In any of the embodiments described inthis paragraph, the UTR can be a 5′-UTR, a 3′-UTR or both 5′-UTR and3′-UTR of a nucleic acid molecule. Table 5 shows exemplary 5′-UTR and3′-UTR sequences that can be used in connection with the presentdisclosure.

TABLE 5 Exemplary Untranslated Region (UTR) Sequences. SEQUENCENAME (SEQ ID AMINO ACID OR NUCLEIC NO:) ACID SEQUENCE 5′-UTR DNAGAAATAAGAGAGAAAAGAAGAGTAA Sequence (SEQ ID GAAGAAATATAAGA NO: 46)5′-UTR RNA GAAAUAAGAGAGAAAAGAAGAGUAA Sequence (SEQ ID GAAGAAAUAUAAGANO: 47) 5′-UTR DNA CTTGTTCTTTTTGCAGAAGCTCAGA Sequence (SEQ IDATAAACGCTCAACTTTGG NO: 48) 5′-UTR RNA CUUGUUCUUUUUGCAGAAGCUCAGASequence (SEQ ID AUAAACGCUCAACUUUGG NO: 49) 5′-UTR DNAGCAGGAGCCAGGGCTGGGCATAAAA Sequence (SEQ ID GTCAGGGCAGAGCCATCTATTGCTTNO: 50) ACATTTGCTTCTGACACAACTGTGT TCACTAGCAACCTCAAACAGACACC 5′-UTR RNAGCAGGAGCCAGGGCUGGGCAUAAAA Sequence (SEQ ID GUCAGGGCAGAGCCAUCUAUUGCUUNO: 51) ACAUUUGCUUCUGACACAACUGUGU UCACUAGCAACCUCAAACAGACACC 3′-UTR DNATAGGCTGGAGCCTCGGTGGCCATGC Sequence (SEQ ID TTCTTGCCCCTTGGGCCTCCCCCCANO: 52) GCCCCTCCTCCCCTTCCTGCACCCG TACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC 3′-UTR RNA UAGGCUGGAGCCUCGGUGGCCAUGC Sequence (SEQ IDUUCUUGCCCCUUGGGCCUCCCCCCA NO: 53) GCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGU CUGAGUGGGCGGC 3′-UTR DNAGCTCGCTTTCTTGCTGTCCAATTTC Sequence (SEQ ID TATTAAAGGTTCCTTTGTTCCCTAANO: 54) GTCCAACTACTAAACTGGGGGATAT TATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTT TCATTGC 3′-UTR RNA GCUCGCUUUCUUGCUGUCCAAUUUCSequence (SEQ ID UAUUAAAGGUUCCUUUGUUCCCUAA NO: 55)GUCCAACUACUAAACUGGGGGAUAU UAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUU UCAUUGC 3′-UTR DNA CTGGTACTGCATGCACGCAATGCTASequence (SEQ ID GCTGCCCCTTTCCCGTCCTGGGTAC NO: 56)CCCGAGTCTCCCCCGACCTCGGGTC CCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTC CAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGC CACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAG TTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCAC ACC 3′ UTR RNA CUGGUACUGCAUGCACGCAAUGCUASequence (SEQ ID GCUGCCCCUUUCCCGUCCUGGGUAC NO: 57)CCCGAGUCUCCCCCGACCUCGGGUC CCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUC CAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGC CACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAG UUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCAC ACC

In specific embodiments, the nucleic acid molecule of the presentdisclose comprises a 5′-UTR selected from SEQ ID NOS:46-51. In specificembodiments, the nucleic acid molecule of the present disclose comprisesa 3′-UTR selected from SEQ ID NOS:52-57. In specific embodiments, thenucleic acid molecule of the present disclose comprises a 5′-UTRselected from SEQ ID NOS:46-51 and a 3′-UTR selected from SEQ IDNOS:52-57. In any of the embodiments described in this paragraph, thenucleic acid molecule may further comprise a coding region having asequence as described in Section 6.3.1, such as any of the DNA codingsequences in Tables 1 to 4 or equivalent RNA sequences thereof. Inparticular embodiments, the nucleic acid molecules described in thisparagraph can be RNA molecules in vitro transcribed.

6.3.4 The Polyadenylation (Poly-A) Regions

During natural RNA processing, a long chain of adenosine nucleotides(poly-A region) is normally added to messenger RNA (mRNA) molecules toincrease the stability of the molecule. Immediately after transcription,the 3′-end of the transcript is cleaved to free a 3′-hydroxy. Thenpoly-A polymerase adds a chain of adenosine nucleotides to the RNA. Theprocess, called polyadenylation, adds a poly-A region that is between100 and 250 residues long. Without being bound by the theory, it iscontemplated that a poly-A region can confer various advantages to thenucleic acid molecule of the present disclosure.

Accordingly, in some embodiments, a nucleic acid molecule of the presentdisclosure (e.g., an mRNA) comprises a polyadenylation signal. In someembodiments, a nucleic acid molecule of the present disclosure (e.g., anmRNA) comprises one or more polyadenylation (poly-A) regions. In someembodiments, a poly-A region is composed entirely of adenine nucleotidesor functional analogs thereof. In some embodiments, the nucleic acidmolecule comprises at least one poly-A region at its 3′-end. In someembodiments, the nucleic acid molecule comprises at least one poly-Aregion at its 5′-end. In some embodiments, the nucleic acid moleculecomprises at least one poly-A region at its 5′-end and at least onepoly-A region at its 3′-end.

According to the present disclosure, the poly-A region can have variedlengths in different embodiments. Particularly, in some embodiments, thepoly-A region of a nucleic acid molecule of the present disclosure is atleast 30 nucleotides in length. In some embodiments, the poly-A regionof a nucleic acid molecule of the present disclosure is at least 35nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 40nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 45nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 50nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 55nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 60nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 65nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 70nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 75nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 80nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 85nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 90nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 95nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 100nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 110nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 120nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 130nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 140nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 150nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 160nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 170nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 180nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 190nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 200nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 225nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 250nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 275nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 300nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 350nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 400nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 450nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 500nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 600nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 700nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 800nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 900nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1000nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1100nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1200nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1300nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1400nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1500nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1600nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1700nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1800nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 1900nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 2000nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 2250nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 2500nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 2750nucleotides in length. In some embodiments, the poly-A region of anucleic acid molecule of the present disclosure is at least 3000nucleotides in length.

In some embodiments, length of a poly-A region in a nucleic acidmolecule can be selected based on the overall length of the nucleic acidmolecule, or a portion thereof (such as the length of the coding regionor the length of an open reading frame of the nucleic acid molecule,etc.). For example, in some embodiments, the poly-A region accounts forabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more of the total length of nucleic acidmolecule containing the poly-A region.

Without being bound by the theory, it is contemplated that certainRNA-binding proteins can bind to the poly-A region located at the 3′-endof an mRNA molecule. These poly-A binding proteins (PABP) can modulatemRNA expression, such as interacting with translation initiationmachinery in a cell and/or protecting the 3′-poly-A tails fromdegradation. Accordingly, in some embodiments, in some embodiments, thenucleic acid molecule of the present disclosure (e.g., mRNA) comprisesat least one binding site for poly-A binding protein (PABP). In otherembodiments, the nucleic acid molecule is conjugated or complex with aPABP before loaded into a delivery vehicle (e.g., lipid nanoparticles).

In some embodiments, the nucleic acid molecule of the present disclosure(e.g., mRNA) comprises a poly-A-G Quartet. The G-quartet is a cyclichydrogen bonded array of four guanosine nucleotides that can be formedby G-rich sequences in both DNA and RNA. In this embodiment, theG-quartet is incorporated at the end of the poly-A region. The resultantpolynucleotides (e.g., mRNA) may be assayed for stability, proteinproduction and other parameters including half-life at various timepoints. It has been discovered that the polyA-G quartet structureresults in protein production equivalent to at least 75% of that seenusing a poly-A region of 120 nucleotides alone.

In some embodiments, the nucleic acid molecule of the present disclosure(e.g., mRNA) may include a poly-A region and may be stabilized by theaddition of a 3′-stabilizing region. In some embodiments, the3′-stabilizing region which may be used to stabilize a nucleic acidmolecule (e.g., mRNA) including the poly-A or poly-A-G Quartetstructures as described in International Patent Publication No.WO2013/103659, the content of which is incorporated herein by referencein its entirety.

In other embodiments, the 3′-stabilizing region which may be used inconnection with the nucleic acid molecules of the present disclosureinclude a chain termination nucleoside such as but is not limited to3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytosine,3′-deoxyguanosine, 3′-deoxythymine, 2′,3′-dideoxynucleosides, such as2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine,2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, oran O-methylnucleoside, 3′-deoxynucleoside, 2′,3′-dideoxynucleoside3′-O-methylnucleosides, 3′-O-ethylnucleosides, 3′-arabinosides, andother alternative nucleosides known in the art and/or described herein.

6.3.5 Secondary Structure

Without being bound by the theory, it is contemplated that a stem-loopstructure can direct RNA folding, protect structural stability of anucleic acid molecule (e.g., mRNA), provide recognition sites for RNAbinding proteins, and serve as a substrate for enzymatic reactions. Forexample, the incorporation of a miR sequence and/or a TEE sequencechanges the shape of the stem loop region which may increase and/ordecrease translation (Kedde et al. A Pumilio-induced RNA structureswitch in p27-3′UTR controls miR-221 and miR-222 accessibility. Nat CellBiol., 2010 October; 12(10):1014-20, the content of which is hereinincorporated by reference in its entirety).

Accordingly, in some embodiments, the nucleic acid molecules asdescribed herein (e.g., mRNA) or a portion thereof may assume astem-loop structure, such as but is not limited to a histone stem loop.In some embodiments, the stem-loop structure is formed from a stem-loopsequence that is about 25 or about 26 nucleotides in length such as, butnot limited to, those as described in International Patent PublicationNo. WO2013/103659, the content of which is incorporated herein byreference in its entirety. Additional examples of stem-loop sequencesinclude those described in International Patent Publication No.WO2012/019780 and International Patent Publication No. WO201502667, thecontents of which are incorporated herein by reference. In someembodiments, the step-loop sequence comprises a TEE as described herein.In some embodiments, the step-loop sequence comprises a miR sequence asdescribed herein. In specific embodiments, the stem loop sequence mayinclude a miR-122 seed sequence. In specific embodiments, the nucleicacid molecule comprises the stem-loop sequence CAAAGGCTCTTTTCAGAGCCACCA(SEQ ID NO:58). In other embodiments, the nucleic acid moleculecomprises the stem-loop sequence CAAAGGCUCUUUUCAGAGCCACCA (SEQ IDNO:59).

In some embodiments, the nucleic acid molecule of the present disclosure(e.g., mRNA) comprises a stem-loop sequence located upstream (to the5′-end) of the coding region in a nucleic acid molecule. In someembodiments, the stem-loop sequence is located within the 5′-UTR of thenucleic acid molecule. In some embodiments, the nucleic acid molecule ofthe present disclosure (e.g., mRNA) comprises a stem-loop sequencelocated downstream (to the 3′-end) of the coding region in a nucleicacid molecule. In some embodiments, the stem-loop sequence is locatedwithin the 3′-UTR of the nucleic acid molecule. In some cases, a nucleicacid molecule can contain more than one stem-loop sequences. In someembodiment, the nucleic acid molecule comprises at least one stem-loopsequence in the 5′-UTR, and at least one stem-loop sequence in the3′-UTR.

In some embodiments, a nucleic acid molecule comprising a stem-loopstructure further comprises a stabilization region. In some embodiment,the stabilization region comprises at least one chain terminatingnucleoside that functions to slow down degradation and thus increasesthe half-life of the nucleic acid molecule. Exemplary chain terminatingnucleoside that can be used in connection with the present disclosureinclude but are not limited to 3′-deoxyadenosine (cordycepin),3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine,2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine,2′,3′-dideoxythymine, a 2′-deoxynucleoside, or an O-methylnucleoside,3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′-O-methylnucleosides,3′-O-ethylnucleosides, 3′-arabinosides, and other alternativenucleosides known in the art and/or described herein. In otherembodiments, a stem-loop structure may be stabilized by an alteration tothe 3′-region of the polynucleotide that can prevent and/or inhibit theaddition of oligio(U) (International Patent Publication No.WO2013/103659, incorporated herein by reference in its entirety).

In some embodiments, a nucleic acid molecule of the present disclosurecomprises at least one stem-loop sequence and a poly-A region orpolyadenylation signal. Non-limiting examples of polynucleotidesequences comprising at least one stem-loop sequence and a poly-A regionor a polyadenylation signal include those described in InternationalPatent Publication No. WO2013/120497, International Patent PublicationNo. WO2013/120629, International Patent Publication No. WO2013/120500,International Patent Publication No. WO2013/120627, International PatentPublication No. WO2013/120498, International Patent Publication No.WO2013/120626, International Patent Publication No. WO2013/120499 andInternational Patent Publication No. WO2013/120628, the content of eachof which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loopsequence and a poly-A region or a polyadenylation signal can encode fora pathogen antigen or fragment thereof such as the polynucleotidesequences described in International Patent Publication No.WO2013/120499 and International Patent Publication No. WO2013/120628,the content of each of which is incorporated herein by reference in itsentirety.

In some embodiments, the nucleic acid molecule comprising a stem-loopsequence and a poly-A region or a polyadenylation signal can encode fora therapeutic protein such as the polynucleotide sequences described inInternational Patent Publication No. WO2013/120497 and InternationalPatent Publication No. WO2013/120629, the content of each of which isincorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loopsequence and a poly-A region or a polyadenylation signal can encode fora tumor antigen or fragment thereof such as the polynucleotide sequencesdescribed in International Patent Publication No. WO2013/120500 andInternational Patent Publication No. WO2013/120627, the content of eachof which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loopsequence and a poly-A region or a polyadenylation signal can code for anallergenic antigen or an autoimmune self-antigen such as thepolynucleotide sequences described in International Patent PublicationNo. WO2013/120498 and International Patent Publication No.WO2013/120626, the content of each of which is incorporated herein byreference in its entirety.

6.3.6 Functional Nucleotide Analogs

In some embodiments, a payload nucleic acid molecule described hereincontains only canonical nucleotides selected from A (adenosine), G(guanosine), C (cytosine), U (uridine), and T (thymidine). Without beingbound by the theory, it is contemplated that certain functionalnucleotide analogs can confer useful properties to a nucleic acidmolecule. Examples of such as useful properties in the context of thepresent disclosure include but are not limited to increased stability ofthe nucleic acid molecule, reduced immunogenicity of the nucleic acidmolecule in inducing innate immune responses, enhanced production ofprotein encoded by the nucleic acid molecule, increased intracellulardelivery and/or retention of the nucleic acid molecule, and/or reducedcellular toxicity of the nucleic acid molecule, etc.

Accordingly, in some embodiments, a payload nucleic acid moleculecomprises at least one functional nucleotide analog as described herein.In some embodiments, the functional nucleotide analog contains at leastone chemical modification to the nucleobase, the sugar group and/or thephosphate group. Accordingly, a payload nucleic acid molecule comprisingat least one functional nucleotide analog contains at least one chemicalmodification to the nucleobases, the sugar groups, and/or theinternucleoside linkage. Exemplary chemical modifications to thenucleobases, sugar groups, or internucleoside linkages of a nucleic acidmolecule are provided herein.

As described herein, ranging from 0% to 100% of all nucleotides in apayload nucleic acid molecule can be functional nucleotide analogs asdescribed herein. For example, in various embodiments, from about 1% toabout 20%, from about 1% to about 25%, from about 1% to about 50%, fromabout 1% to about 60%, from about 1% to about 70%, from about 1% toabout 80%, from about 1% to about 90%, from about 1% to about 95%, fromabout 10% to about 20%, from about 10% to about 25%, from about 10% toabout 50%, from about 10% to about 60%, from about 10% to about 70%,from about 10% to about 80%, from about 10% to about 90%, from about 10%to about 95%, from about 10% to about 100%, from about 20% to about 25%,from about 20% to about 50%, from about 20% to about 60%, from about 20%to about 70%, from about 20% to about 80%, from about 20% to about 90%,from about 20% to about 95%, from about 20% to about 100%, from about50% to about 60%, from about 50% to about 70%, from about 50% to about80%, from about 50% to about 90%, from about 50% to about 95%, fromabout 50% to about 100%, from about 70% to about 80%, from about 70% toabout 90%, from about 70% to about 95%, from about 70% to about 100%,from about 80% to about 90%, from about 80% to about 95%, from about 80%to about 100%, from about 90% to about 95%, from about 90% to about100%, or from about 95% to about 100% of all nucleotides in a nucleicacid molecule are functional nucleotide analogs described herein. In anyof these embodiments, a functional nucleotide analog can be present atany position(s) of a nucleic acid molecule, including the 5′-terminus,3′-terminus, and/or one or more internal positions. In some embodiments,a single nucleic acid molecule can contain different sugarmodifications, different nucleobase modifications, and/or differenttypes internucleoside linkages (e.g., backbone structures).

As described herein, ranging from 0% to 100% of all nucleotides of akind (e.g., all purine-containing nucleotides as a kind, or allpyrimidine-containing nucleotides as a kind, or all A, G, C, T or U as akind) in a payload nucleic acid molecule can be functional nucleotideanalogs as described herein. For example, in various embodiments, fromabout 1% to about 20%, from about 1% to about 25%, from about 1% toabout 50%, from about 1% to about 60%, from about 1% to about 70%, fromabout 1% to about 80%, from about 1% to about 90%, from about 1% toabout 95%, from about 10% to about 20%, from about 10% to about 25%,from about 10% to about 50%, from about 10% to about 60%, from about 10%to about 70%, from about 10% to about 80%, from about 10% to about 90%,from about 10% to about 95%, from about 10% to about 100%, from about20% to about 25%, from about 20% to about 50%, from about 20% to about60%, from about 20% to about 70%, from about 20% to about 80%, fromabout 20% to about 90%, from about 20% to about 95%, from about 20% toabout 100%, from about 50% to about 60%, from about 50% to about 70%,from about 50% to about 80%, from about 50% to about 90%, from about 50%to about 95%, from about 50% to about 100%, from about 70% to about 80%,from about 70% to about 90%, from about 70% to about 95%, from about 70%to about 100%, from about 80% to about 90%, from about 80% to about 95%,from about 80% to about 100%, from about 90% to about 95%, from about90% to about 100%, or from about 95% to about 100% of a kind ofnucleotides in a nucleic acid molecule are functional nucleotide analogsdescribed herein. In any of these embodiments, a functional nucleotideanalog can be present at any position(s) of a nucleic acid molecule,including the 5′-terminus, 3′-terminus, and/or one or more internalpositions. In some embodiments, a single nucleic acid molecule cancontain different sugar modifications, different nucleobasemodifications, and/or different types internucleoside linkages (e.g.,backbone structures).

6.3.7 Modification to Nucleobases

In some embodiments, a functional nucleotide analog contains anon-canonical nucleobase. In some embodiments, canonical nucleobases(e.g., adenine, guanine, uracil, thymine, and cytosine) in a nucleotidecan be modified or replaced to provide one or more functional analogs ofthe nucleotide. Exemplary modification to nucleobases include but arenot limited to one or more substitutions or modifications including butnot limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and/or thiosubstitutions; one or more fused or open rings, oxidation, and/orreduction.

In some embodiments, the non-canonical nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having an modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil,6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s²U), 4-thio-uracil(s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil(ho⁵U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or5-bromo-uracil), 3-methyl-uracil (m³U), 5-methoxy-uracil (mo⁵U), uracil5-oxyacetic acid (cmo⁵U), uracil 5-oxyacetic acid methyl ester (mcmo⁵U),5-carboxymethyl-uracil (cm⁵U), 1-carboxymethyl-pseudouridine,5-carboxyhydroxymethyl-uracil (chm⁵U), 5-carboxyhydroxymethyl-uracilmethyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uracil (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uracil (mcm⁵s²U),5-aminomethyl-2-thio-uracil (nm⁵s²U), 5-methylaminomethyl-uracil(mnm⁵U), 5-methylaminomethyl-2-thio-uracil (mnm⁵s²U),5-methylaminomethyl-2-seleno-uracil (mnm⁵se²U), 5-carbamoylmethyl-uracil(ncm⁵U), 5-carboxymethylaminomethyl-uracil (cmnm⁵U),5-carboxymethylaminomethyl-2-thio-uracil (cmnm⁵s²U), 5-propynyl-uracil,1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm⁵U),1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil(τm⁵5s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m⁵U, i.e., havingthe nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),1-ethyl-pseudouridine (Et¹ψ), 5-methyl-2-thio-uracil (m⁵s²U),1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine,3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil,5-methyl-dihydrouracil (m⁵D), 2-thio-dihydrouracil,2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uracil (m⁵U),5-(isopentenylaminomethyl)-2-thio-uracil (m⁵s²U),5,2′-O-dimethyl-uridine (m⁵Um), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uracil,deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil,5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil,5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil,5-methoxy-2-thio-uracil, and 5-[3-(1-E-propenylamino)]uracil.

In some embodiments, the non-canonical nucleobase is a modifiedcytosine. Exemplary nucleobases and nucleosides having a modifiedcytosine include 5-aza-cytosine, G aza-cytosine, pseudoisocytidine,3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine(f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C),5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine(hmSC), 1-methyl-pseudoisocytidine, pyrrolo-cytosine,pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C),2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), 5,2′-O-dimethyl-cytidine (mSCm),N4-acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl-cytidine(m4Cm), 5-formyl-2′-O-methyl-cytidine (fSCm),N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine,5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and5-(2-azidoethyl)-cytosine.

In some embodiments, the non-canonical nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having an alternative adenineinclude 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine,7-deaza-8-azaadenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenine (m1A),2-methyl-adenine (m2A), N6-methyl-adenine (m6A),2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A),2-methylthio-N6-isopentenyl-adenine (ms2i6A),N6-(cis-hydroxyisopentenyl)adenine (io6A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A),N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A),N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A),2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A),N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine(hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A),N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, N6,2′-O-dimethyl-adenosine (m6Am),N6,N6,2′-O-trimethyl-adenosine (m62Am), 1,2′-O-dimethyl-adenosine(mlAm), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine,N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine,N6-formyl-adenine, and N6-hydroxymethyl-adenine.

In some embodiments, the non-canonical nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine(preQ1), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine,6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine(m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine,1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine(m22G), N2,7-dimethyl-guanine (m2,7G), N2, N2,7-dimethyl-guanine(m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine,1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine,N2,N2-dimethyl-6-thio-guanine, N2-methyl-2′-O-methyl-guanosine (m2Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm),1-methyl-2′-O-methyl-guanosine (m1Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m1Im), 1-thio-guanine, and 0-6-methyl-guanine.

In some embodiments, the non-canonical nucleobase of a functionalnucleotide analog can be independently a purine, a pyrimidine, a purineor pyrimidine analog. For example, in some embodiments, thenon-canonical nucleobase can be modified adenine, cytosine, guanine,uracil, or hypoxanthine. In other embodiments, the non-canonicalnucleobase can also include, for example, naturally-occurring andsynthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; or 1,3,5 triazine.

6.3.8 Modification to the Sugar

In some embodiments, a functional nucleotide analog contains anon-canonical sugar group. In various embodiments, the non-canonicalsugar group can be a 5-carbon or 6-carbon sugar (such as pentose,ribose, arabinose, xylose, glucose, galactose, or a deoxy derivativethereof) with one or more substitutions, such as a halo group, a hydroxygroup, a thiol group, an alkyl group, an alkoxy group, an alkenyloxygroup, an alkynyloxy group, an cycloalkyl group, an aminoalkoxy group,an alkoxyalkoxy group, an hydroxyalkoxy group, an amino group, an azidogroup, an aryl group, an aminoalkyl group, an aminoalkenyl group, anaminoalkynyl group, etc.

Generally, RNA molecules contains the ribose sugar group, which is a5-membered ring having an oxygen. Exemplary, non-limiting alternativenucleotides include replacement of the oxygen in ribose (e.g., with S,Se, or alkylene, such as methylene or ethylene); addition of a doublebond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino (that also has a phosphoramidate backbone)); multicyclicforms (e.g., tricyclo and “unlocked” forms, such as glycol nucleic acid(GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol unitsattached to phosphodiester bonds), threose nucleic acid (TNA, whereribose is replace with α-L-threofuranosyl-(3′→2′)), and peptide nucleicacid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone).

In some embodiments, the sugar group contains one or more carbons thatpossess the opposite stereochemical configuration of the correspondingcarbon in ribose. Thus, a nucleic acid molecule can include nucleotidescontaining, e.g., arabinose or L-ribose, as the sugar. In someembodiments, the nucleic acid molecule includes at least one nucleosidewherein the sugar is L-ribose, 2′-O-methyl-ribose, 2′-fluoro-ribose,arabinose, hexitol, an LNA, or a PNA.

6.3.9 Modifications to the Internucleoside Linkage

In some embodiments, the payload nucleic acid molecule of the presentdisclosure can contain one or more modified internucleoside linkage(e.g., phosphate backbone). Backbone phosphate groups can be altered byreplacing one or more of the oxygen atoms with a different substituent.

In some embodiments, the functional nucleotide analogs can include thereplacement of an unaltered phosphate moiety with anotherinternucleoside linkage as described herein. Examples of alternativephosphate groups include, but are not limited to, phosphorothioate,phosphoroselenates, boranophosphates, boranophosphate esters, hydrogenphosphonates, phosphoramidates, phosphorodiamidates, alkyl or arylphosphonates, and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bealtered by the replacement of a linking oxygen with nitrogen (bridgedphosphoramidates), sulfur (bridged phosphorothioates), and carbon(bridged methylene-phosphonates).

The alternative nucleosides and nucleotides can include the replacementof one or more of the non-bridging oxygens with a borane moiety (BH₃),sulfur (thio), methyl, ethyl, and/or methoxy. As a non-limiting example,two non-bridging oxygens at the same position (e.g., the alpha (a), beta(β) or gamma (γ) position) can be replaced with a sulfur (thio) and amethoxy. The replacement of one or more of the oxygen atoms at theposition of the phosphate moiety (e.g., α-thio phosphate) is provided toconfer stability (such as against exonucleases and endonucleases) to RNAand DNA through the unnatural phosphorothioate backbone linkages.Phosphorothioate DNA and RNA have increased nuclease resistance andsubsequently a longer half-life in a cellular environment.

Other internucleoside linkages that may be employed according to thepresent disclosure, including internucleoside linkages which do notcontain a phosphorous atom, are described herein.

Additional examples of nucleic acid molecules (e.g., mRNA),compositions, formulations and/or methods associated therewith that canbe used in connection with the present disclosure further include thosedescribed in WO2002/098443, WO2003/051401, WO2008/052770, WO2009127230,WO2006122828, WO2008/083949, WO2010088927, WO2010/037539, WO2004/004743,WO2005/016376, WO2006/024518, WO2007/095976, WO2008/014979,WO2008/077592, WO2009/030481, WO2009/095226, WO2011069586, WO2011026641,WO2011/144358, WO2012019780, WO2012013326, WO2012089338, WO2012113513,WO2012116811, WO2012116810, WO2013113502, WO2013113501, WO2013113736,WO2013143698, WO2013143699, WO2013143700, WO2013/120626, WO2013120627,WO2013120628, WO2013120629, WO2013174409, WO2014127917, WO2015/024669,WO2015/024668, WO2015/024667, WO2015/024665, WO2015/024666,WO2015/024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738,WO2015101416, the content of each of which is incorporated herein in itsentirety.

Therapeutic nucleic acid molecules as described herein can by isolatedor synthesized using methods known in the art. In some embodiments, DNAor RNA molecules to be used in connection with the present disclosureare chemically synthesized. In other embodiments, DNA or RNA moleculesto be used in connection with the present disclosure are isolated from anatural source.

In some embodiments, mRNA molecules to be used in connection with thepresent disclosure are biosynthesized using a host cell. In particularembodiments, an mRNA is produced by transcribing a corresponding DNAsequencing using a host cell. In some embodiments, a DNA sequenceencoding an mRNA sequence is incorporated into an expression vector,which vector is then introduced into a host cell (e.g., E. coli) usingmethods known in the art. The host cell is then cultured under asuitable condition to produce mRNA transcripts. Other methods forproducing an mRNA molecule from an encoding DNA are known in the art.For example, in some embodiments, a cell-free (in vitro) transcriptionsystem comprising enzymes of the transcription machinery of a host cellcan be used to produce mRNA transcripts. An exemplary cell-freetranscription reaction system is described in Example 1 of the presentdisclosure.

6.4 Nanoparticle Compositions

In one aspect, nucleic acid molecules described herein are formulatedfor in vitro and in vivo delivery. Particularly, in some embodiments thenucleic acid molecule is formulated into a lipid-containing composition.In some embodiments, the lipid-containing composition forms lipidnanoparticles enclosing the nucleic acid molecule within a lipid shell.In some embodiments, the lipid shells protects the nucleic acidmolecules from degradation. In some embodiments, the lipid nanoparticlesalso facilitate transportation of the enclosed nucleic acid moleculesinto intracellular compartments and/or machinery to exert an intendedtherapeutic of prophylactic function. In certain embodiments, nucleicacids, when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipid nanoparticles comprisingnucleic acids and their method of preparation are known in the art, suchas those disclosed in, e.g., U.S. Patent Publication No. 2004/0142025,U.S. Patent Publication No. 2007/0042031, PCT Publication No. WO2017/004143, PCT Publication No.¶ WO 2015/199952, PCT Publication No. WO2013/016058, and PCT Publication No. WO 2013/086373, the fulldisclosures of each of which are herein incorporated by reference intheir entirety for all purposes.

In some embodiments, the largest dimension of a nanoparticle compositionprovided herein is 1 μm or shorter (e.g., ≤1 μm, ≤900 nm, ≤800 nm, ≤700nm, ≤600 nm, ≤500 nm, ≤400 nm, ≤300 nm, ≤200 nm, ≤175 nm, ≤150 nm, ≤125nm, ≤100 nm, ≤75 nm, ≤50 nm, or shorter), such as when measured bydynamic light scattering (DLS), transmission electron microscopy,scanning electron microscopy, or another method. In one embodiment, thelipid nanoparticle provided herein has at least one dimension that is inthe range of from about 40 to about 200 nm. In one embodiment, the atleast one dimension is in the range of from about 40 to about 100 nm.

Nanoparticle compositions that can be used in connection with thepresent disclosure include, for example, lipid nanoparticles (LNPs),nano liproprotein particles, liposomes, lipid vesicles, and lipoplexes.In some embodiments, nanoparticle compositions are vesicles includingone or more lipid bilayers. In some embodiments, a nanoparticlecomposition includes two or more concentric bilayers separated byaqueous compartments. Lipid bilayers may be functionalized and/orcrosslinked to one another. Lipid bilayers may include one or moreligands, proteins, or channels.

In some embodiments, nanoparticle compositions as described comprise alipid component including at least one lipid, such as a compoundaccording to one of Formulae (I) to (IV) (and sub-formulas thereof) asdescribed herein. For example, in some embodiments, a nanoparticlecomposition may include a lipid component including one of compoundsprovided herein. Nanoparticle compositions may also include one or moreother lipid or non-lipid components as described below.

6.4.1 Cationic Lipids

In some embodiments, the lipid-containing composition comprises at leastone lipid compound according to Formula (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or        C₂-C₁₂ alkenylene, wherein one or more —CH₂— in the alkylene or        alkenylene is optionally replaced by —O—;    -   L¹ is —OC(═O)R¹, —C(═O)OR¹, —OC(═O)OR¹, —C(═O)R¹, —OR¹,        —S(O)XR¹, —S—SR¹,        —C(═O)SR¹—SC(═O)R¹—NR^(a)C(═O)R¹—C(═O)NR^(b)R^(c), —NR^(b)R^(c),        —OC(═O)NR^(b)R^(c), —NR^(a)C(═O)OR¹, —SC(═S)R¹, —C(═S)SR¹,        —C(═S)R¹, —CH(OH)R¹, —P(═O)(OR^(b))(OR^(c)), —(C₆-C₁₀        arylene)-R¹, -(6- to 10-membered heteroarylene)-R¹, or R¹;    -   L² is —OC(═O)R², —C(═O)OR², —OC(═O)OR², —C(═O)R², —OR²,        —S(O)XR², —S—SR², —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R²,        —C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f),        —NR^(d)C(═O)OR², —SC(═S)R², —C(═S)SR², —C(═S)R², —CH(OH)R²,        —P(═O)(OR^(e))(OR^(f)), —(C₆-C₁₀ arylene)-R², -(6- to        10-membered heteroarylene)-R², or R²;    -   R¹ and R² are each independently C₆-C₃₂ alkyl or C₆-C₃₂ alkenyl;    -   R^(a), R^(b), R^(d), and R^(e) are each independently H, C₁-C₂₄        alkyl, or C₂-C₂₄ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₃₂ alkyl or C₂-C₃₂        alkenyl;    -   G³ is C₂-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₃-C₈ cycloalkylene,        or C₃-C₈ cycloalkenylene;    -   R³ is —N(R⁴)R⁵;    -   R⁴ is C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 4- to 8-membered        heterocyclyl, or C₆-C₁₀ aryl; or R⁴, G³ or part of G³, together        with the nitrogen to which they are attached form a cyclic        moiety;    -   R⁵ is C₁-C₁₂ alkyl or C₃-C₈ cycloalkyl; or R⁴, R⁵, together with        the nitrogen to which they are attached form a cyclic moiety;    -   x is 0, 1 or 2; and    -   wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl,        heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene,        cycloalkenylene, arylene, heteroarylene, and cyclic moiety is        independently optionally substituted.

In one embodiment, provided herein is a compound of Formula (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or        C₂-C₁₂ alkenylene;    -   L¹ is —OC(═O)R¹, —C(═O)OR¹, —OC(═O)OR¹, —C(═O)R¹, —OR¹,        —S(O)_(x)R¹, —S—SR¹, —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹,        —C(═O)NR^(b)R^(c), —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c),        —NR^(a)C(═O)OR¹, —SC(═S)R¹, —C(═S)SR¹, —C(═S)R¹, —CH(OH)R¹,        —P(═O)(OR^(b))(OR^(c)), —(C₆-C₁₀ arylene)-R¹, -(6- to        10-membered heteroarylene)-R¹, or R¹;    -   L² is —OC(═O)R², —C(═O)OR², —OC(═O)OR², —C(═O)R², —OR²,        —S(O)XR², —S—SR², —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R²,        —C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f),        —NR^(d)C(═O)OR², —SC(═S)R², —C(═S)SR², —C(═S)R², —CH(OH)R²,        —P(═O)(OR^(e))(OR^(f)), —(C₆-C₁₀ arylene)-R², -(6- to        10-membered heteroarylene)-R², or R²;    -   R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;    -   R^(a), R^(b), R^(d), and W are each independently H, C₁-C₁₂        alkyl, or C₂-C₁₂ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂        alkenyl;    -   G³ is C₂-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₃-C₈ cycloalkylene,        or C₃-C₈ cycloalkenylene;    -   R³ is —N(R⁴)R⁵;    -   R⁴ is C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or C₆-C₁₀ aryl;    -   R⁵ is C₁-C₁₂ alkyl;    -   x is 0, 1 or 2; and    -   wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl,        alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene,        and heteroarylene is independently optionally substituted.

In one embodiment, provided herein is a compound of Formula (II):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   is a single bond or a double bond;    -   G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or        C₂-C₁₂ alkenylene, wherein one or more —CH₂— in the alkylene or        alkenylene is optionally replaced by —O—;    -   L¹ is —OC(═O)R¹, —C(═O)OR¹, —OC(═O)OR¹, —C(═O)R¹, —OR¹,        —S(O)XR¹, —S—SR¹, —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹,        —C(═O)NR^(b)R^(c), —NR^(b)R^(c)—OC(═O)NR^(b)R^(c),        —NR^(a)C(═O)OR¹, —SC(═S)R¹, —C(═S)SR¹, —C(═S)R¹, —CH(OH)R¹,        —P(═O)(OR^(b))(OR^(c)), —(C₆-C₁₀ arylene)-R¹, -(6- to        10-membered heteroarylene)-R¹, or R¹;    -   L² is —OC(═O)R², —C(═O)OR², —OC(═O)OR², —C(═O)R², —OR²,        —S(O)XR², —S—SR², —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R²,        —C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f),        —NR^(d)C(═O)OR², —SC(═S)R², —C(═S)SR², —C(═S)R², —CH(OH)R²,        —P(═O)(OR^(e))(OR^(f)), —(C₆-C₁₀ arylene)-R², -(6- to        10-membered heteroarylene)-R², or R²;    -   R¹ and R² are each independently C₆-C₃₂ alkyl or C₆-C₃₂ alkenyl;    -   R^(a), R^(b), R^(d), and R^(e) are each independently H, C₁-C₂₄        alkyl, or C₂-C₂₄ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₃₂ alkyl or C₂-C₃₂        alkenyl;    -   G⁴ is a bond, C₁-C₂₃ alkylene, C₂-C₂₃ alkenylene, C₃-C₈        cycloalkylene, or C₃-C₈ cycloalkenylene;    -   R³ is —N(R⁴)R⁵;    -   R⁴ is C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 4- to        8-membered heterocyclyl, or C₆-C₁₀ aryl; or R⁴, G³ or part of        G³, together with the nitrogen to which they are attached form a        cyclic moiety;    -   R⁵ is C₁-C₁₂ alkyl or C₃-C₈ cycloalkyl; or R⁴, R⁵, together with        the nitrogen to which they are attached form a cyclic moiety;    -   x is 0, 1 or 2; and    -   wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl,        heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene,        cycloalkenylene, arylene, heteroarylene, and cyclic moiety is        independently optionally substituted.

In one embodiment, provided herein is a compound of Formula (II):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   is a single bond or a double bond;    -   G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or        C₂-C₁₂ alkenylene;    -   L¹ is —OC(═O)R¹, —C(═O)OR¹, —OC(═O)OR¹, —C(═O)R¹, —OR¹,        —S(O)XR¹, —S—SR¹, —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹,        —C(═O)NR^(b)R^(c), —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c),        —NR^(a)C(═O)OR¹, —SC(═S)R¹, —C(═S)SR¹, —C(═S)R¹, —CH(OH)R¹,        —P(═O)(OR^(b))(OR^(c)), —(C₆-C₁₀ arylene)-R¹, -(6- to        10-membered heteroarylene)-R¹, or R¹;    -   L² is —OC(═O)R², —C(═O)OR², —OC(═O)OR², —C(═O)R², —OR²,        —S(O)XR², —S—SR², —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R²,        —C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f),        —NR^(d)C(═O)OR², —SC(═S)R², —C(═S)SR², —C(═S)R², —CH(OH)R²,        —P(═O)(OR^(e))(OR^(f)), —(C₆-C₁₀ arylene)-R², -(6- to        10-membered heteroarylene)-R², or R²;    -   R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;    -   R^(a), R^(b), R^(d), and R^(e) are each independently H, C₁-C₁₂        alkyl, or C₂-C₁₂ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂        alkenyl;    -   G⁴ is a bond, C₁-C₂₃ alkylene, C₂-C₂₃ alkenylene, C₃-C₈        cycloalkylene, or C₃-C₈ cycloalkenylene;    -   R³ is —N(R⁴)R⁵;    -   R⁴ is C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or        C₆-C₁₀ aryl;    -   R⁵ is C₁-C₁₂ alkyl;    -   x is 0, 1 or 2; and    -   wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl,        alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene,        and heteroarylene is independently optionally substituted.

In one embodiment,

is a single bond. In one embodiment,

is a double bond. In one embodiment,

is a double bond, and the compound has a (Z)-configuration. In oneembodiment,

is a double bond, and the compound has a (E)-configuration.

In one embodiment, provided herein is a compound of Formula (III):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, provided herein is a compound of Formula (IV):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G¹ is a bond. In one embodiment, G² is a bond. In oneembodiment, G¹ and G² are both a bond.

In one embodiment, G¹ and G² are each independently C₂-C₁₂ alkylene orC₂-C₁₂ alkenylene. In one embodiment, G¹ and G² are each independentlyC₂-C₁₂ alkylene. In one embodiment, G¹ and G² are each independentlyC₂-C₁₂ alkenylene. In one embodiment, G¹ and G² are each independentlyC₃-C₇ alkylene. In one embodiment, G¹ and G² are each independently C₅alkylene.

In one embodiment, G¹ is unsubstituted. In one embodiment, G¹ issubstituted. In one embodiment, G¹ is substituted with —OH. In oneembodiment, G¹ is substituted with (a second) L¹ (i.e., G¹ is connectedto two L¹). In one embodiment, G¹ is substituted with —O—(C₆-C₂₄ alkyl).In one embodiment, G¹ is substituted with —O—(C₆-C₂₄ alkenyl). In oneembodiment, G¹ is substituted with —C(═O)—(C₆-C₂₄ alkyl). In oneembodiment, G¹ is substituted with —C(═O)—(C₆-C₂₄ alkenyl).

In one embodiment, G² is unsubstituted. In one embodiment, G² issubstituted. In one embodiment, G² is substituted with —OH. In oneembodiment, G² is substituted with (a second) L² (i.e., G² is connectedto two L²). In one embodiment, G² is substituted with —O—(C₆-C₂₄ alkyl).In one embodiment, G² is substituted with —O—(C₆-C₂₄ alkenyl). In oneembodiment, G² is substituted with —C(═O)—(C₆-C₂₄ alkyl). In oneembodiment, G² is substituted with —C(═O)—(C₆-C₂₄ alkenyl).

In one embodiment, one or more —CH₂— in the alkylene or alkenylene in G¹and/or G² is optionally replaced by —O—. In one embodiment, G¹ and G²are each independently C₅-C₉ alkylene, wherein one or more —CH₂— in thealkylene is optionally replaced by —O—. In one embodiment, G¹ and G² areeach independently C₅-C₇ alkylene, wherein one or more —CH₂— in thealkylene is optionally replaced by —O—. In one embodiment, G¹ and G² areboth —CH₂—CH₂—O—CH₂—CH₂—. In one embodiment, G¹ and G² are both—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—.

In one embodiment, the compound is a compound of Formula (I-A)

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (II-A):

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (III-A):

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (IV-A):

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, y and z are each independently an integer from 2 to10. In one embodiment, y and z are each independently an integer from 2to 6. In one embodiment, y and z are each independently an integer from4 to 10.

In one embodiment, y and z are different. In one embodiment, y and z arethe same. In one embodiment, y and z are the same and are selected from4, 5, 6, 7, 8, and 9. In one embodiment, y is 5 and z is 5.

In one embodiment, L¹ is —OC(═O)R¹, —C(═O)OR¹, —OC(═O)OR¹, —C(═O)R¹,—OR¹, —S(O)_(x)R¹, —S—SR¹, —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹,—C(═O)NR^(b)R^(c), —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c),—NR^(a)C(═O)OR¹, —SC(═S)R¹, —C(═S)SR¹, —C(═S)R¹, —CH(OH)R¹, or—P(═O)(OR^(b))(OR^(c)). In one embodiment, L¹ is —(C₆-C₁₀ arylene)-R¹.In one embodiment, L¹ is -(6- to 10-membered heteroarylene)-R¹. In oneembodiment, L¹ is R¹.

In one embodiment, L¹ is —OC(═O)R¹, —C(═O)OR¹, —C(═O)SR¹, —SC(═O)R¹,—NR^(a)C(═O)R¹, or —C(═O)NR^(b)R^(c). In one embodiment, L¹ is—OC(═O)R¹, —C(═O)OR¹, —NR^(a)C(═O)R¹, or —C(═O)NR^(b)R^(c). In oneembodiment, L¹ is —OC(═O)R¹. In one embodiment, L¹ is —C(═O)OR¹. In oneembodiment, L¹ is —NR^(a)C(═O)R¹. In one embodiment, L¹ is—C(═O)NR^(b)R^(c). In one embodiment, L¹ is —NR^(a)C(═O)NR^(b)R^(c). Inone embodiment, L¹ is —OC(═O)NR^(b)R^(c). In one embodiment, L¹ is—NR^(a)C(═O)OR¹,

In one embodiment, L² is —OC(═O)R², —C(═O)OR², —OC(═O)OR², —C(═O)R²,—OR², —S(O)_(x)R², —S—SR², —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R²,—C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f),—NR^(d)C(═O)OR², —SC(═S)R², —C(═S)SR², —C(═S)R², —CH(OH)R², or—P(═O)(OR^(e))(OR^(f)). In one embodiment, L² is —(C₆-C₁₀ arylene)-R².In one embodiment, L² is -(6- to 10-membered heteroarylene)-R². In oneembodiment, L² is R².

In one embodiment, L² is —OC(═O)R², —C(═O)OR², —C(═O)SR², —SC(═O)R²,—NR^(d)C(═O)R², or —C(═O)NR^(e)R^(f). In one embodiment, L² is—OC(═O)R², —C(═O)OR², —NR^(d)C(═O)R², or —C(═O)NR^(e)R^(f). In oneembodiment, L² is —OC(═O)R². In one embodiment, L² is —C(═O)OR². In oneembodiment, L² is —NR^(d)C(═O)R². In one embodiment, L² is—C(═O)NR^(e)R^(f). In one embodiment, L² is —NR^(d)C(═O)NR^(e)R^(f). Inone embodiment, L² is —OC(═O)NR^(e)R^(f). In one embodiment, L² is—NR^(d)C(═O)OR².

In one embodiment, L¹ is —OC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)OR¹, or—C(═O)NR^(b)R^(c) and L² is —OC(═O)R², —NR^(d)C(═O)R², —C(═O)OR², or—C(═O)NR^(e)R^(f). In one embodiment, L¹ is —OC(═O)R¹, —C(═O)OR¹, or—C(═O)NR^(b)R^(c) and L² is —OC(═O)R², —C(═O)OR², or —C(═O)NR^(e)R^(f).In one embodiment, L¹ is —OC(═O)R¹ and L² is —OC(═O)R². In oneembodiment, L¹ is —OC(═O)R¹ and L² is —NR^(d)C(═O)R². In one embodiment,L¹ is —NR^(a)C(═O)R¹ and L² is —NR^(d)C(═O)R². In one embodiment, L¹ is—C(═O)OR¹ and L² is —C(═O)OR². In one embodiment, L¹ is —C(═O)OR¹ and L²is —C(═O)NR^(e)R^(f). In one embodiment, L¹ is —C(═O)NR^(b)R^(c) and L²is —C(═O)NR^(e)R^(f).

In one embodiment, L¹ is —NR^(a)C(═O)NR^(b)R^(c) and L² is—NR^(d)C(═O)NR^(e)R^(f). In one embodiment, L¹ is —OC(═O)NR^(b)R^(c) andL² is —OC(═O)NR^(e)R^(f). In one embodiment, L¹ is —NR^(a)C(═O)OR¹ andL² is —NR^(d)C(═O)OR².

In one embodiment, the compound is a compound of Formula (I-B), (I-B′),(I-B″), (I C), (I-D), or (I-E):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (II-B),(II-B′), (II-B″), (II-C), (II-D), or (II-E):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (III-B),(III-B′), (III-B″), (III-C), (III-D), or (III-E):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (IV-B),(IV-B′), (IV-B″), (IV-C), (IV-D), or (IV-E):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (I-F), (I-F′),(I-F″), (I-G), (I-H), or (I-I):

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (II-F),(II-F′), (II-F″), (II-G), (II-H), or (II-I):

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (III-F),(III-F′), (III-F″), (III-G), (III-H), or (III-I):

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (IV-F),(IV-F′), (IV-F″), (IV-G), (IV-H), or (IV-I):

-   -   wherein y and z are each independently an integer from 2 to 12,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, y and z are each independently an integer from 2 to10. In one embodiment, y and z are each independently an integer from 2to 6. In one embodiment, y and z are each independently an integer from4 to 10.

In one embodiment, y and z are different. In one embodiment, y and z arethe same. In one embodiment, y and z are the same and are selected from4, 5, 6, 7, 8, and 9. In one embodiment, y is 5 and z is 5.

In one embodiment, G³ is C₂-C₂₄ alkylene. In one embodiment, G³ isC₂-C₁₂ alkylene. In one embodiment, G³ is C₂-C₈ alkylene. In oneembodiment, G³ is C₂-C₆ alkylene. In one embodiment, G³ is C₂-C₄alkylene. In one embodiment, G³ is C₂ alkylene.

In one embodiment, G³ is C₄ alkylene.

In one embodiment, G³ is substituted with one or more oxo. In oneembodiment, G³ is —(C₁-C₂₃ alkylene)-C(═O)—. In one embodiment, G³ is—(C₁-C₁₁ alkylene)-C(═O)—. In one embodiment, G³ is —(C₁-C₇alkylene)-C(═O)—. In one embodiment, G³ is —(C₁-C₅ alkylene)-C(═O)—. Inone embodiment, G³ is —(C₁-C₃ alkylene)-C(═O)—. In one embodiment, G³ is—CH₂—C(═O)—. In one embodiment, G³ is —CH₂—CH₂—CH₂—C(═O)—. In oneembodiment, the —C(═O)— is connected to the nitrogen atom, and thealkylene is connected to R³.

In one embodiment, the compound is a compound of Formula (I-J), (I-J′),(I-J″), (I-K), (I-L), or (I-M):

-   -   wherein y and z are each independently an integer from 2 to 12,        and    -   s is an integer from 2 to 24,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, y and z are each independently an integer from 2 to10. In one embodiment, y and z are each independently an integer from 2to 6. In one embodiment, y and z are each independently an integer from4 to 10.

In one embodiment, y and z are different. In one embodiment, y and z arethe same. In one embodiment, y and z are the same and are selected from4, 5, 6, 7, 8, and 9. In one embodiment, y is 5 and z is 5.

In one embodiment, s is an integer from 2 to 12. In one embodiment, s isan integer from 2 to 8. In one embodiment, s is an integer from 2 to 6.In one embodiment, s is an integer from 2 to 4. In one embodiment, s is2. In one embodiment, s is 4.

In one embodiment, y is 5, z is 5, and s is 2.

In one embodiment, y is 5, z is 5, and s is 4.

In one embodiment, G³ is C₂-C₂₄ alkenylene. In one embodiment, G³ isC₂-C₁₂ alkenylene. In one embodiment, G³ is C₂-C₈ alkenylene. In oneembodiment, G³ is C₂-C₆ alkenylene. In one embodiment, G³ is C₂-C₄alkenylene.

In one embodiment, G³ is C₃-C₈ cycloalkylene. In one embodiment, G³ isC₅-C₆ cycloalkylene.

In one embodiment, G³ is C₃-C₈ cycloalkenylene. In one embodiment, G³ isC₅-C₆ cycloalkenylene.

In one embodiment, G⁴ is a bond.

In one embodiment, G⁴ is C₁-C₂₃ alkylene. In one embodiment, G⁴ isC₁-C₁₁ alkylene. In one embodiment, G⁴ is C₁-C₇ alkylene. In oneembodiment, G⁴ is C₁-C₅ alkylene. In one embodiment, G⁴ is C₁-C₃alkylene. In one embodiment, G⁴ is C₁ alkylene. In one embodiment, G⁴ isC₂ alkylene. In one embodiment, G⁴ is C₃ alkylene. In one embodiment, G⁴is C₄ alkylene.

In one embodiment, the compound is a compound of Formula (II-J),(II-J′), (II-J″), (II-K), (II-L), or (II-M):

-   -   wherein y and z are each independently an integer from 2 to 12,        and    -   u is an integer from 0 to 23,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (III-J),(III-J′), (III-J″), (III-K), (III-L), or (III-M):

-   -   wherein y and z are each independently an integer from 2 to 12,        and    -   u is an integer from 0 to 23,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (IV-J),(IV-J′), (IV-J″), (IV-K), (IV-L), or (IV-M):

-   -   wherein y and z are each independently an integer from 2 to 12,        and    -   u is an integer from 0 to 23,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, y and z are each independently an integer from 2 to10. In one embodiment, y and z are each independently an integer from 2to 6. In one embodiment, y and z are each independently an integer from4 to 10.

In one embodiment, y and z are different. In one embodiment, y and z arethe same. In one embodiment, y and z are the same and are selected from4, 5, 6, 7, 8, and 9. In one embodiment, y is 5 and z is 5.

In one embodiment, u is an integer from 0 to 12. In one embodiment, u isan integer from 0 to 8. In one embodiment, u is an integer from 0 to 6.In one embodiment, u is an integer from 0 to 4. In one embodiment, u is0. In one embodiment, u is 1. In one embodiment, u is 2. In oneembodiment, u is 3. In one embodiment, u is 4.

In one embodiment, y is 5, z is 5, and u is 0.

In one embodiment, y is 5, z is 5, and u is 2.

In one embodiment, G⁴ is C₂-C₂₃ alkenylene. In one embodiment, G⁴ isC₂-C₁₂ alkenylene. In one embodiment, G⁴ is C₂-C₈ alkenylene. In oneembodiment, G⁴ is C₂-C₆ alkenylene. In one embodiment, G⁴ is C₂-C₄alkenylene.

In one embodiment, G⁴ is C₃-C₈ cycloalkylene. In one embodiment, G⁴ isC₅-C₆ cycloalkylene.

In one embodiment, G⁴ is C₃-C₈ cycloalkenylene. In one embodiment, G⁴ isC₅-C₆ cycloalkenylene.

In one embodiment, R⁵ is C₁-C₁₂ alkyl. In one embodiment, R⁵ is C₁-C₁₀alkyl. In one embodiment, R⁵ is C₁-C₈ alkyl. In one embodiment, R⁵ isC₁-C₆ alkyl. In one embodiment, R⁵ is C₁-C₄ alkyl. In one embodiment, R⁵is C₁-C₂ alkyl. In one embodiment, R⁵ is methyl. In one embodiment, R⁵is ethyl. In one embodiment, R⁵ is propyl. In one embodiment, R⁵ isn-butyl. In one embodiment, R⁵ is n-hexyl. In one embodiment, R⁵ isn-octyl. In one embodiment, R⁵ is n-nonyl.

In one embodiment, R⁵ is C₃-C₈ cycloalkyl. In one embodiment, R⁵ iscyclopropyl. In one embodiment, R⁵ is cyclobutyl. In one embodiment, R⁵is cyclopentyl. In one embodiment, R⁵ is cyclohexyl. In one embodiment,R⁵ is cycloheptyl. In one embodiment, R⁵ is cyclooctyl.

In one embodiment, R⁴, R⁵, together with the nitrogen to which they areattached form a cyclic moiety.

In one embodiment, the cyclic moiety (formed by R⁴ and R⁵ together withthe nitrogen to which they are attached) is heterocyclyl. In oneembodiment, the cyclic moiety is heterocycloalkyl. In one embodiment,the cyclic moiety is 4- to 8-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 4-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 5-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 6-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 7-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 8-membered heterocycloalkyl.

In one embodiment, the cyclic moiety (formed by R⁴ and R⁵ together withthe nitrogen to which they are attached) is azetidin-1-yl. In oneembodiment, the cyclic moiety is pyrrolidin-1-yl. In one embodiment, thecyclic moiety is piperidin-1-yl. In one embodiment, the cyclic moiety isazepan-1-yl. In one embodiment, the cyclic moiety is azocan-1-yl. In oneembodiment, the cyclic moiety is morpholinyl. In one embodiment, thecyclic moiety is piperazin-1-yl. The point of attachment in these groupsis to G³.

As described herein and unless otherwise specified, the substitutionpatterns for R⁵ also applies to the cyclic moiety formed by R⁴ and R⁵together with the nitrogen to which they are attached.

In one embodiment, R⁵ is unsubstituted.

In one embodiment, R⁵ is substituted with one or more substituentsselected from the group consisting of oxo, —OR^(g), —NR^(g)C(═O)R^(h),—C(═O)NR^(g)R^(h), —C(═O)R^(h), —OC(═O)R^(h), —C(═O)OR^(h) and —O—R—OH,wherein:

-   -   R^(g) is at each occurrence independently H or C₁-C₆ alkyl;    -   R^(h) is at each occurrence independently C₁-C₆ alkyl; and    -   R^(i) is at each occurrence independently C₁-C₆ alkylene.

In one embodiment, R⁵ is substituted with one or more hydroxyl. In oneembodiment, R⁵ is substituted with one hydroxyl.

In one embodiment, R⁵ is substituted with one or more hydroxyl and oneor more oxo. In one embodiment, R⁵ is substituted with one hydroxyl andone oxo. In one embodiment, R⁵ is —CH₂CH₂OH.

In one embodiment, R⁵ is —(CH₂)_(p)Q, —(CH₂)_(p)CHQR, —CHQR, or —CQ(R)₂,wherein Q is C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkynyl,4- to 8-membered heterocyclyl, C₆-C₁₀ aryl, 5- to 10-memberedheteroaryl, —OR, —O(CH₂)_(p)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,—CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —N(R)R²², —O(CH₂)_(p)OR, —N(R)C(═NR²³)N(R)₂,—N(R)C(═CHR²³)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R,—N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂,—N(OR)C(═NR²³)N(R)₂, —N(OR)C(═CHR²³)N(R)₂, —C(═NR²³)N(R)₂, —C(═NR²³)R,—C(O)N(R)OR, or —C(R)N(R)₂C(O)OR, and each p is independently 1, 2, 3,4, or 5;

-   -   R²² is C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₃-C₈ cycloalkynyl,        4- to 8-membered heterocyclyl, C₆-C₁₀ aryl, or 5- to 10-membered        heteroaryl;    -   R²³ is H, —CN, —NO₂, C₁-C₆ alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂,        C₂-C₆ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₃-C₈        cycloalkynyl, 4- to 8-membered heterocyclyl, C₆-C₁₀ aryl, or 5-        to 10-membered heteroaryl;    -   each R is independently H, C₁-C₃ alkyl, or C₂-C₃ alkenyl; or two        R in a N(R)₂ moiety together with the nitrogen to which they are        attached form a cyclic moiety; and    -   each X is independently F, Cl, Br, or I.

In one embodiment, the compound is a compound of Formula (I-N), (I-N′),(I-N″), (I-O), (I-P), or (I-Q):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   s is an integer from 2 to 24,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (II-N),(II-N′), (II-N″), (II-O), (II-P), or (II-Q):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   u is an integer from 0 to 23,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (III-N),(III-N′), (III-N″), (III-O), (III-P), or (III-Q):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   u is an integer from 0 to 23,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (IV-N),(IV-N′), (IV-N″), (IV-O), (IV-P), or (IV-Q):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   u is an integer from 0 to 23,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (I-R), (I-R′),(I-R″), (I-S), (I-T), or (I-U):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   s is an integer from 2 to 24,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (II-R),(II-R′), (II-R″), (II-S), (II-T), or (II-U):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   u is an integer from 0 to 23,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (III-R),(III-R′), (III-R″) (III-S), (III-T) or (III-U):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   u is an integer from 0 to 23,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, the compound is a compound of Formula (IV-R),(IV-R′), (IV-R″), (IV-S), (IV-T), or (IV-U):

-   -   wherein y and z are each independently an integer from 2 to 12,    -   u is an integer from 0 to 23,    -   t is an integer from 1 to 12, and    -   R⁶ is hydrogen or hydroxyl,        or a pharmaceutically acceptable salt, prodrug or stereoisomer        thereof.

In one embodiment, y and z are each independently an integer from 2 to10. In one embodiment, y and z are each independently an integer from 2to 6. In one embodiment, y and z are each independently an integer from4 to 10.

In one embodiment, y and z are different. In one embodiment, y and z arethe same. In one embodiment, y and z are the same and are selected from4, 5, 6, 7, 8, and 9. In one embodiment, y is 5 and z is 5.

In one embodiment, s is an integer from 2 to 12. In one embodiment, s isan integer from 2 to 8. In one embodiment, s is an integer from 2 to 6.In one embodiment, s is an integer from 2 to 4. In one embodiment, s is2. In one embodiment, s is 4.

In one embodiment, y is 5, z is 5, and s is 2.

In one embodiment, y is 5, z is 5, and s is 4.

In one embodiment, u is an integer from 0 to 12. In one embodiment, u isan integer from 0 to 8. In one embodiment, u is an integer from 0 to 6.In one embodiment, u is an integer from 0 to 4. In one embodiment, u is0. In one embodiment, u is 1. In one embodiment, u is 2.

In one embodiment, u is 3. In one embodiment, u is 4.

In one embodiment, y is 5, z is 5, and u is 0.

In one embodiment, y is 5, z is 5, and u is 2.

In one embodiment, t is an integer from 1 to 10. In one embodiment, t isan integer from 1 to 8. In one embodiment, t is an integer from 1 to 6.In one embodiment, t is an integer from 1 to 4. In one embodiment, t isan integer from 1 to 3. In one embodiment, t is an integer from 1 to 2.In one embodiment, t is 1. In one embodiment, t is 2. In one embodiment,t is 3. In one embodiment, t is 4. In one embodiment, t is 5. In oneembodiment, t is 6. In one embodiment, t is 7.

In one embodiment, R⁴ is C₁-C₁₂ alkyl. In one embodiment, R⁴ is C₁-C₈alkyl. In one embodiment, R⁴ is C₁-C₆ alkyl. In one embodiment, R⁴ isC₁-C₄ alkyl. In one embodiment, R⁴ is methyl. In one embodiment, R⁴ isethyl. In one embodiment, R⁴ is n-propyl. In one embodiment, R⁴ isn-butyl. In one embodiment, R⁴ is n-pentyl. In one embodiment, R⁴ isn-hexyl. In one embodiment, R⁴ is n-octyl. In one embodiment, R⁴ isn-nonyl.

In one embodiment, R⁴ is C₃-C₈ cycloalkyl. In one embodiment, R⁴ iscyclopropyl. In one embodiment, R⁴ is cyclobutyl. In one embodiment, R⁴is cyclopentyl. In one embodiment, R⁴ is cyclohexyl. In one embodiment,R⁴ is cycloheptyl. In one embodiment, R⁴ is cyclooctyl.

In one embodiment, R⁴ is C₃-C₈ cycloalkenyl. In one embodiment, R⁴ iscyclopropenyl. In one embodiment, R⁴ is cyclobutenyl. In one embodiment,R⁴ is cyclopentenyl. In one embodiment, R⁴ is cyclohexenyl. In oneembodiment, R⁴ is cycloheptenyl. In one embodiment, R⁴ is cyclooctenyl.

In one embodiment, R⁴ is C₆-C₁₀ aryl. In one embodiment, R⁴ is phenyl.

In one embodiment, R⁴ is 4- to 8-membered heterocyclyl. In oneembodiment, R⁴ is 4- to 8-membered heterocycloalkyl. In one embodiment,R⁴ is oxetanyl. In one embodiment, R⁴ is tetrahydrofuranyl. In oneembodiment, R⁴ is tetrahydropyranyl. In one embodiment, R⁴ istetrahydrothiopyranyl. In one embodiment, R⁴ is N-methylpiperidinyl.

In one embodiment, R⁴, G³ or part of G³, together with the nitrogen towhich they are attached form a cyclic moiety.

In one embodiment, the cyclic moiety (formed by R⁴, G³ or part of G³,together with the nitrogen to which they are attached) is heterocyclyl.In one embodiment, the cyclic moiety is heterocycloalkyl. In oneembodiment, the cyclic moiety is 4- to 8-membered heterocycloalkyl. Inone embodiment, the cyclic moiety is 4-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 5-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 6-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 7-membered heterocycloalkyl. In oneembodiment, the cyclic moiety is 8-membered heterocycloalkyl.

In one embodiment, the cyclic moiety (formed by R⁴, G³ or part of G³,together with the nitrogen to which they are attached) is azetidin-3-yl.In one embodiment, the cyclic moiety is pyrrolidin-3-yl. In oneembodiment, the cyclic moiety is piperidin-4-yl. In one embodiment, thecyclic moiety is azepan-4-yl. In one embodiment, the cyclic moiety isazocan-5-yl. The point of attachment for these groups is to thedirection of the nitrogen that is connected to G¹ and G².

As described herein and unless otherwise specified, the substitutionpatterns for R⁴ also applies to the cyclic moiety formed by R⁴, G³ orpart of G³, together with the nitrogen to which they are attached.

In one embodiment, R⁴ is unsubstituted.

In one embodiment, R⁴ is substituted with one or more substituentsselected from the group consisting of oxo, —OR^(g), —NR^(g)C(═O)R^(h),—C(═O)NR^(g)R^(h), —C(═O)R^(h), —OC(═O)R^(h), —C(═O)OR^(h) and —O—R—OH,wherein:

-   -   R^(g) is at each occurrence independently H or C₁-C₆ alkyl;    -   R^(h) is at each occurrence independently C₁-C₆ alkyl; and    -   R^(i) is at each occurrence independently C₁-C₆ alkylene.

In one embodiment, R⁴ is substituted with one or more hydroxyl. In oneembodiment, R⁴ is substituted with one hydroxyl.

In one embodiment, R⁴ is substituted with one or more hydroxyl and oneor more oxo. In one embodiment, R⁴ is substituted with one hydroxyl andone oxo.

In one embodiment, R³ has one of the following structures:

In one embodiment, R³ has the structure of

In one embodiment, R³ has the structure of

In one embodiment, R¹ and R² are each independently branched C₆-C₃₂alkyl or branched C₆-C₃₂ alkenyl. In one embodiment, R¹ and R² are eachindependently branched C₆-C₂₄ alkyl or branched C₆-C₂₄ alkenyl.

In one embodiment, R¹ and R² are each independently —R⁷—CH(R⁸)(R⁹),wherein R⁷ is C₁-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀alkyl or C₂-C₁₀ alkenyl.

In one embodiment, R¹ is straight C₆-C₃₂ alkyl. In one embodiment, R¹ isstraight C₆-C₂₄ alkyl. In one embodiment, R¹ is straight C₇-C₁₅ alkyl.In one embodiment, R¹ is straight C₇ alkyl. In one embodiment, R¹ isstraight C₈ alkyl. In one embodiment, R¹ is straight C₉ alkyl. In oneembodiment, R¹ is straight C₁₀ alkyl. In one embodiment, R¹ is straightC₁₁ alkyl. In one embodiment, R¹ is straight C₁₂ alkyl. In oneembodiment, R¹ is straight C₁₃ alkyl. In one embodiment, R¹ is straightC₁₄ alkyl. In one embodiment, R¹ is straight C₁₅ alkyl.

In one embodiment, R¹ is straight C₆-C₃₂ alkenyl. In one embodiment, R¹is straight C₆-C₂₄ alkenyl. In one embodiment, R¹ is straight C₇-C₁₇alkenyl. In one embodiment, R¹ is straight C₇ alkenyl. In oneembodiment, R¹ is straight C₈ alkenyl. In one embodiment, R¹ is straightC₉ alkenyl. In one embodiment, R¹ is straight C₁₀ alkenyl. In oneembodiment, R¹ is straight C₁₁ alkenyl. In one embodiment, R¹ isstraight C₁₂ alkenyl. In one embodiment, R¹ is straight C₁₃ alkenyl. Inone embodiment, R¹ is straight C₁₄ alkenyl. In one embodiment, R¹ isstraight C₁₅ alkenyl. In one embodiment, R¹ is straight C₁₆ alkenyl. Inone embodiment, R¹ is straight C₁₇ alkenyl.

In one embodiment, R¹ is branched C₆-C₃₂ alkyl. In one embodiment, R¹ isbranched C₆-C₂₄ alkyl. In one embodiment, R¹ is —R⁷—CH(R⁸)(R⁹), whereinR⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl. Inone embodiment, R¹ is —R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₁ alkylene, andR⁸ and R⁹ are independently C₄-C₈ alkyl.

In one embodiment, R¹ is branched C₆-C₃₂ alkenyl. In one embodiment, R¹is branched C₆-C₂₄ alkenyl. In one embodiment, R¹ is —R⁷—CH(R⁸)(R⁹),wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀alkenyl. In one embodiment, R¹ is —R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₁alkylene, and R⁸ and R⁹ are independently C₆-C₁₀ alkenyl.

In one embodiment, R² is straight C₆-C₃₂ alkyl. In one embodiment, R² isstraight C₆-C₂₄ alkyl. In one embodiment, R² is straight C₇-C₁₅ alkyl.In one embodiment, R² is straight C₇ alkyl. In one embodiment, R² isstraight C₈ alkyl. In one embodiment, R² is straight C₉ alkyl. In oneembodiment, R² is straight C₁₀ alkyl. In one embodiment, R² is straightC₁₁ alkyl. In one embodiment, R² is straight C₁₂ alkyl. In oneembodiment, R² is straight C₁₃ alkyl. In one embodiment, R² is straightC₁₄ alkyl. In one embodiment, R² is straight C₁₅ alkyl.

In one embodiment, R² is straight C₆-C₃₂ alkenyl. In one embodiment, R²is straight C₆-C₂₄ alkenyl. In one embodiment, R² is straight C₇-C₁₇alkenyl. In one embodiment, R² is straight C₇ alkenyl. In oneembodiment, R² is straight C₈ alkenyl. In one embodiment, R² is straightC₉ alkenyl. In one embodiment, R² is straight C₁₀ alkenyl. In oneembodiment, R² is straight C₁₁ alkenyl. In one embodiment, R² isstraight C₁₂ alkenyl. In one embodiment, R² is straight C₁₃ alkenyl. Inone embodiment, R² is straight C₁₄ alkenyl. In one embodiment, R² isstraight C₁₅ alkenyl. In one embodiment, R² is straight C₁₆ alkenyl. Inone embodiment, R² is straight C₁₇ alkenyl.

In one embodiment, R² is branched C₆-C₃₂ alkyl. In one embodiment, R² isbranched C₆-C₂₄ alkyl. In one embodiment, R² is —R⁷—CH(R⁸)(R⁹), whereinR⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl. Inone embodiment, R² is —R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₁ alkylene, andR⁸ and R⁹ are independently C₄-C₈ alkyl.

In one embodiment, R² is branched C₆-C₃₂ alkenyl. In one embodiment, R²is branched C₆-C₂₄ alkenyl. In one embodiment, R² is —R⁷—CH(R⁸)(R⁹),wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀alkenyl. In one embodiment, R² is —R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₁alkylene, and R⁸ and R⁹ are independently C₆-C₁₀ alkenyl.

In one embodiment, R^(c) is straight C₆-C₃₂ alkyl. In one embodiment,R^(c) is straight C₆-C₂₄ alkyl. In one embodiment, R^(c) is straightC₇-C₁₅ alkyl. In one embodiment, R^(c) is straight C₇ alkyl. In oneembodiment, R^(c) is straight C₈ alkyl. In one embodiment, R^(c) isstraight C₉ alkyl. In one embodiment, R^(c) is straight C₁₀ alkyl. Inone embodiment, R^(c) is straight C₁₁ alkyl. In one embodiment, R^(c) isstraight C₁₂ alkyl. In one embodiment, R^(c) is straight C₁₃ alkyl. Inone embodiment, R^(c) is straight C₁₄ alkyl. In one embodiment, R^(c) isstraight C₁₅ alkyl.

In one embodiment, R^(c) is straight C₆-C₃₂ alkenyl. In one embodiment,R^(c) is straight C₆-C₂₄ alkenyl. In one embodiment, R^(c) is straightC₇-C₁₇ alkenyl. In one embodiment, R^(c) is straight C₇ alkenyl. In oneembodiment, R^(c) is straight C₈ alkenyl. In one embodiment, R^(c) isstraight C₉ alkenyl. In one embodiment, R^(c) is straight C₁₀ alkenyl.In one embodiment, R^(c) is straight C₁₁ alkenyl. In one embodiment,R^(c) is straight C₁₂ alkenyl. In one embodiment, R^(c) is straight C₁₃alkenyl. In one embodiment, R^(c) is straight C₁₄ alkenyl. In oneembodiment, R^(c) is straight C₁₅ alkenyl. In one embodiment, R^(c) isstraight C₁₆ alkenyl. In one embodiment, R^(c) is straight C₁₇ alkenyl.

In one embodiment, R^(c) is branched C₆-C₃₂ alkyl. In one embodiment,R^(c) is branched C₆-C₂₄ alkyl. In one embodiment, R^(c) is—R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ areindependently C₂-C₁₀ alkyl. In one embodiment, R^(c) is —R⁷—CH(R⁸)(R⁹),wherein R⁷ is C₀-C₁ alkylene, and R⁸ and R⁹ are independently C₄-C₈alkyl.

In one embodiment, R^(c) is branched C₆-C₃₂ alkenyl. In one embodiment,R^(c) is branched C₆-C₂₄ alkenyl. In one embodiment, R^(c) is—R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ areindependently C₂-C₁₀ alkenyl. In one embodiment, R^(c) is—R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₁ alkylene, and R⁸ and R⁹ areindependently C₆-C₁₀ alkenyl.

In one embodiment, R^(f) is straight C₆-C₃₂ alkyl. In one embodiment,R^(f) is straight C₆-C₂₄ alkyl. In one embodiment, R^(f) is straightC₇-C₁₅ alkyl. In one embodiment, R^(f) is straight C₇ alkyl. In oneembodiment, R^(f) is straight C₈ alkyl. In one embodiment, R^(f) isstraight C₉ alkyl. In one embodiment, R^(f) is straight C₁₀ alkyl. Inone embodiment, R^(f) is straight C₁₁ alkyl. In one embodiment, R^(f) isstraight C₁₂ alkyl. In one embodiment, R^(f) is straight C₁₃ alkyl. Inone embodiment, R^(f) is straight C₁₄ alkyl. In one embodiment, R^(f) isstraight C₁₅ alkyl.

In one embodiment, R^(f) is straight C₆-C₃₂ alkenyl. In one embodiment,R^(f) is straight C₆-C₂₄ alkenyl. In one embodiment, R^(f) is straightC₇-C₁₇ alkenyl. In one embodiment, R^(f) is straight C₇ alkenyl. In oneembodiment, R^(f) is straight C₈ alkenyl. In one embodiment, R^(f) isstraight C₉ alkenyl. In one embodiment, R^(f) is straight C₁₀ alkenyl.In one embodiment, R^(f) is straight C₁₁ alkenyl. In one embodiment,R^(f) is straight C₁₂ alkenyl. In one embodiment, R^(f) is straight C₁₃alkenyl. In one embodiment, R^(f) is straight C₁₄ alkenyl. In oneembodiment, R^(f) is straight C₁₅ alkenyl. In one embodiment, R^(f) isstraight C₁₆ alkenyl. In one embodiment, R^(f) is straight C₁₇ alkenyl.

In one embodiment, R^(f) is branched C₆-C₃₂ alkyl. In one embodiment,R^(f) is branched C₆-C₂₄ alkyl. In one embodiment, R^(f) is—R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ areindependently C₂-C₁₀ alkyl. In one embodiment, R^(f) is —R⁷—CH(R⁸)(R⁹),wherein R⁷ is C₀-C₁ alkylene, and R⁸ and R⁹ are independently C₄-C₈alkyl.

In one embodiment, R^(f) is branched C₆-C₃₂ alkenyl. In one embodiment,R^(f) is branched C₆-C₂₄ alkenyl. In one embodiment, R^(f) is—R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ areindependently C₂-C₁₀ alkenyl. In one embodiment, R^(f) is—R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₀-C₁ alkylene, and R⁸ and R⁹ areindependently C₆-C₁₀ alkenyl.

In one embodiment, R¹, R², R^(c), and R^(f) are each independentlystraight C₆-C₁₈ alkyl, straight C₆-C₁₈ alkenyl, or —R⁷—CH(R⁸)(R⁹),wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀alkyl or C₂-C₁₀ alkenyl.

In one embodiment, R¹, R², R^(c), and R^(f) are each independentlystraight C₇-C₁₅ alkyl, straight C₇-C₁₅ alkenyl, or —R⁷—CH(R⁸)(R⁹),wherein R⁷ is C₀-C₁ alkylene, and R⁸ and R⁹ are independently C₄-C₈alkyl or C₆-C₁₀ alkenyl.

In one embodiment, R¹, R², R^(c), and R^(f) are each independently oneof the following structures:

In one embodiment, R¹, R², R^(c), and R^(f) are each independentlyoptionally substituted. In one embodiment, the optional substituent is—O—(C₆-C₂₄ alkyl). In one embodiment, the optional substituent is—O—(C₆-C₂₄ alkenyl). In one embodiment, the optional substituent is—C(═O)—(C₆-C₂₄ alkyl). In one embodiment, the optional substituent is—C(═O)—(C₆-C₂₄ alkenyl).

In one embodiment, R^(a) and R^(d) are each independently H. In oneembodiment, R^(a), R^(b), R^(d), and Re are each independently H. In oneembodiment, Ra and R^(d) are each independently C₁-C₂₄ alkyl. In oneembodiment, Ra and Rd are each independently C₁-C₁₈ alkyl. In oneembodiment, R^(a) and R^(d) are each independently C₁-C₁₂ alkyl. In oneembodiment, Ra and Rd are each independently C₁-C₆ alkyl.

In one embodiment, R^(b), R^(c), R^(e), and R^(f) are each independentlyn-hexyl or n-octyl.

In one embodiment, R^(c) and R^(f) are each independently branchedC₆-C₂₄ alkyl or branched C₆-C₂₄ alkenyl. In one embodiment, R^(c) andR^(f) are each independently —R⁷—CH(R⁸)(R⁹), wherein R⁷ is C₁-C₅alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl or C₂-C₁₀alkenyl.

In one embodiment, the compound is a compound in Table 1, or apharmaceutically acceptable salt, prodrug or stereoisomer thereof.

TABLE 1

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 20

Compound 21

Compound 22

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

Compound 35

Compound 36

Compound 37

Compound 38

Compound 39

Compound 40

Compound 41

Compound 42

Compound 43

Compound 44

Compound 45

Compound 46

Compound 47

Compound 48

Compound 49

Compound 50

Compound 51

Compound 52

Compound 54

Compound 55

Compound 56

Compound 57

Compound 58

Compound 59

Compound 60

Compound 61

Compound 62

Compound 63

Compound 64

Compound 65

Compound 66

Compound 67

Compound 68

Compound 69

Compound 70

Compound 71

Compound 72

Compound 73

Compound 74

Compound 75

Compound 76

Compound 77

Compound 78

Compound 79

Compound 80

Compound 81

Compound 82

Compound 83

Compound 84

Compound 85

Compound 86

Compound 87

Compound 88

Compound 89

Compound 90

Compound 91

Compound 92

Compound 93

Compound 94

Compound 95

Compound 96

Compound 97

Compound 98

Compound 99

Compound 101

Compound 102

Compound 103

Compound 104

Compound 105

Compound 106

Compound 107

Compound 108

Compound 109

Compound 110

Compound 111

Compound 112

Compound 113

Compound 114

Compound 115

Compound 116

Compound 117

Compound 118

Compound 119

Compound 120

Compound 121

Compound 122

Compound 123

Compound 124

Compound 125

Compound 126

Compound 127

Compound 128

Compound 129

Compound 130

Compound 131

Compound 132

Compound 133

Compound 134

Compound 135

Compound 136

Compound 137

Compound 138

Compound 139

Compound 140

Compound 141

Compound 142

Compound 143

Compound 144

Compound 145

Compound 146

Compound 147

Compound 148

Compound 149

Compound 150

Compound 151

Compound 152

Compound 153

Compound 154

Compound 155

It is understood that any embodiment of the compounds provided herein,as set forth above, and any specific substituent and/or variable in thecompound provided herein, as set forth above, may be independentlycombined with other embodiments and/or substituents and/or variables ofthe compounds to form embodiments not specifically set forth above. Inaddition, in the event that a list of substituents and/or variables islisted for any particular group or variable, it is understood that eachindividual substituent and/or variable may be deleted from theparticular embodiment and/or claim and that the remaining list ofsubstituents and/or variables will be considered to be within the scopeof embodiments provided herein.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

6.4.2 Other Ionizable Lipids

As described herein, in some embodiments, a nanoparticle compositionprovided herein comprises one or more charged or ionizable lipids inaddition to a lipid according Formulae (I) to (IV) (and sub-formulasthereof). Without being bound by the theory, it is contemplated thatcertain charged or zwitterionic lipid components of a nanoparticlecomposition resembles the lipid component in the cell membrane, therebycan improve cellular uptake of the nanoparticle. Exemplary charged orionizable lipids that can form part of the present nanoparticlecomposition include but are not limited to3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)),(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)),(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-den-1-amine,N,N-dimethyl-1-{(1S,2R)-2-octylcyclopropyl}heptadecan-8-amine.Additional exemplary charged or ionizable lipids that can form part ofthe present nanoparticle composition include the lipids (e.g., lipid 5)described in Sabnis et al. “A Novel Amino Lipid Series for mRNADelivery: Improved Endosomal Escape and Sustained Pharmacology andSafety in Non-human Primates”, Molecular Therapy Vol. 26 No 6, 2018, theentirety of which is incorporated herein by reference.

In some embodiments, suitable cationic lipids includeN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP);1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC);1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC);1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC);1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1);N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide(MVLS); dioctadecylamido-glycylspermine (DOGS);3b-[N—(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol);dioctadecyldimethylammonium bromide (DDAB); SAINT-2,N-methyl-4-(dioleyl)methylpyridinium;1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE);1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE);1,2-dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium chloride (DORI);di-alkylated amino acid (DILA²) (e.g., C18:1-norArg-C16);dioleyldimethylammonium chloride (DODAC);1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC);1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC);(R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride(DODAPen-Cl); (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride(DOPen-G); and (R)—N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminiumchloride (DOTAPen). Also suitable are cationic lipids with headgroupsthat are charged at physiological pH, such as primary amines (e.g.,DODAG N′,N′-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide) andguanidinium head groups (e.g., bis-guanidinium-spermidine-cholesterol(BGSC), bis-guanidiniumtren-cholesterol (BGTC), PONA, and(R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G)). Yetanother suitable cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-Cl). In certain embodiments, thecationic lipid is a particular enantiomer or the racemic form, andincludes the various salt forms of a cationic lipid as above (e.g.,chloride or sulfate). For example, in some embodiments, the cationiclipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride(DOTAP-Cl) or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniumsulfate (DOTAP-Sulfate). In some embodiments, the cationic lipid is anionizable cationic lipid such as, e.g., dioctadecyldimethylammoniumbromide (DDAB); 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA);2,2-dilinoleyl-4-(2dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA);heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA); 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP);1,2-dioleyloxy-3-dimethylaminopropane (DODMA); and morpholinocholesterol(Mo-CHOL). In certain embodiments, a lipid nanoparticle includes acombination or two or more cationic lipids (e.g., two or more cationiclipids as above).

Additionally, in some embodiments, the charged or ionizable lipid thatcan form part of the present nanoparticle composition is a lipidincluding a cyclic amine group. Additional cationic lipids that aresuitable for the formulations and methods disclosed herein include thosedescribed in WO2015199952, WO2016176330, and WO2015011633, the entirecontents of each of which are hereby incorporated by reference in theirentireties. Additionally, in some embodiments, the charged or ionizablelipid that can form part of the present nanoparticle composition is alipid including a cyclic amine group. Additional cationic lipids thatare suitable for the formulations and methods disclosed herein includethose described in WO2015199952, WO2016176330, and WO2015011633, theentire contents of each of which are hereby incorporated by reference intheir entireties.

6.4.3 Polymer Conjugated Lipids

In some embodiments, the lipid component of a nanoparticle compositioncan include one or more polymer conjugated lipids, such as PEGylatedlipids (PEG lipids). Without being bound by the theory, it iscontemplated that a polymer conjugated lipid component in a nanoparticlecomposition can improve of colloidal stability and/or reduce proteinabsorption of the nanoparticles. Exemplary cationic lipids that can beused in connection with the present disclosure include but are notlimited to PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, Ceramide-PEG2000, orChol-PEG2000.

In one embodiment, the polymer conjugated lipid is a pegylated lipid.For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asco-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

In one embodiment, the polymer conjugated lipid is present in aconcentration ranging from 1.0 to 2.5 molar percent. In one embodiment,the polymer conjugated lipid is present in a concentration of about 1.7molar percent. In one embodiment, the polymer conjugated lipid ispresent in a concentration of about 1.5 molar percent.

In one embodiment, the molar ratio of cationic lipid to the polymerconjugated lipid ranges from about 35:1 to about 25:1. In oneembodiment, the molar ratio of cationic lipid to polymer conjugatedlipid ranges from about 100:1 to about 20:1.

In one embodiment, the molar ratio of cationic lipid to the polymerconjugated lipid ranges from about 35:1 to about 25:1. In oneembodiment, the molar ratio of cationic lipid to polymer conjugatedlipid ranges from about 100:1 to about 20:1.

In one embodiment, the pegylated lipid has the following Formula:

-   -   or a pharmaceutically acceptable salt, tautomer or stereoisomer        thereof, wherein:    -   R¹² and R¹³ are each independently a straight or branched,        saturated or unsaturated alkyl chain containing from 10 to 30        carbon atoms, wherein the alkyl chain is optionally interrupted        by one or more ester bonds; and    -   w has a mean value ranging from 30 to 60.

In one embodiment, R¹² and R¹³ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average w ranges from 42 to 55, for example, theaverage w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55.In some specific embodiments, the average w is about 49.

In one embodiment, the pegylated lipid has the following Formula:

-   -   wherein the average w is about 49.

6.4.4 Structural Lipids

In some embodiments, the lipid component of a nanoparticle compositioncan include one or more structural lipids. Without being bound by thetheory, it is contemplated that structural lipids can stabilize theamphiphilic structure of a nanoparticle, such as but not limited to thelipid bilayer structure of a nanoparticle. Exemplary structural lipidsthat can be used in connection with the present disclosure include butare not limited to cholesterol, fecosterol, sitosterol, ergosterol,campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolicacid, alpha-tocopherol, and mixtures thereof. In certain embodiments,the structural lipid is cholesterol. In some embodiments, the structurallipid includes cholesterol and a corticosteroid (such as prednisolone,dexamethasone, prednisone, and hydrocortisone), or a combinationthereof.

In one embodiment, the lipid nanoparticles provided herein comprise asteroid or steroid analogue. In one embodiment, the steroid or steroidanalogue is cholesterol. In one embodiment, the steroid is present in aconcentration ranging from 39 to 49 molar percent, 40 to 46 molarpercent, from 40 to 44 molar percent, from 40 to 42 molar percent, from42 to 44 molar percent, or from 44 to 46 molar percent. In oneembodiment, the steroid is present in a concentration of 40, 41, 42, 43,44, 45, or 46 molar percent.

In one embodiment, the molar ratio of cationic lipid to the steroidranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In oneembodiment, the molar ratio of cationic lipid to cholesterol ranges fromabout 5:1 to 1:1. In one embodiment, the steroid is present in aconcentration ranging from 32 to 40 mol percent of the steroid.

In one embodiment, the molar ratio of cationic lipid to the steroidranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In oneembodiment, the molar ratio of cationic lipid to cholesterol ranges fromabout 5:1 to 1:1. In one embodiment, the steroid is present in aconcentration ranging from 32 to 40 mol percent of the steroid.

6.4.5 Phospholipids

In some embodiments, the lipid component of a nanoparticle compositioncan include one or more phospholipids, such as one or more(poly)unsaturated lipids. Without being bound by the theory, it iscontemplated that phospholipids may assemble into one or more lipidbilayers structures. Exemplary phospholipids that can form part of thepresent nanoparticle composition include but are not limited to1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),and sphingomyelin. In certain embodiments, a nanoparticle compositionincludes DSPC. In certain embodiments, a nanoparticle compositionincludes DOPE. In some embodiments, a nanoparticle composition includesboth DSPC and DOPE.

Additional exemplary neutral lipids include, for example,dipalmitoylphosphatidylglycerol (DPPG),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE). In one embodiment, the neutral lipid is1,2-distearoyl-sn-glycero-3phosphocholine (DSPC). In one embodiment, theneutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE andSM.

In one embodiment, the neutral lipid is phosphatidylcholine (PC),phosphatidylethanolamine (PE) phosphatidylserine (PS), phosphatidic acid(PA), or phosphatidylglycerol (PG).

Additionally phospholipids that can form part of the presentnanoparticle composition also include those described in WO2017/112865,the entire content of which is hereby incorporated by reference in itsentirety.

6.4.6 Formulation

According to the present disclosure, nanoparticle compositions describedherein can include at least one lipid component and one or moreadditional components, such as a therapeutic and/or prophylactic agent(e.g., the therapeutic nucleic acid described herein). A nanoparticlecomposition may be designed for one or more specific applications ortargets. The elements of a nanoparticle composition may be selectedbased on a particular application or target, and/or based on theefficacy, toxicity, expense, ease of use, availability, or other featureof one or more elements. Similarly, the particular formulation of ananoparticle composition may be selected for a particular application ortarget according to, for example, the efficacy and toxicity ofparticular combinations of elements.

The lipid component of a nanoparticle composition may include, forexample, a lipid according to one of formulae (I) to (IV) (andsub-formulas thereof) described herein, a phospholipid (such as anunsaturated lipid, e.g., DOPE or DSPC), a PEG lipid, and a structurallipid. The elements of the lipid component may be provided in specificfractions.

In one embodiment, provided herein is a nanoparticle compositionscomprising a cationic or ionizable lipid compound provided herein, atherapeutic agent, and one or more excipients. In one embodiment,cationic or ionizable lipid compound comprises a compound according toone of Formulae (I) to (IV) (and sub-formulas thereof) as describedherein, and optionally one or more additional ionizable lipid compounds.In one embodiment, the one or more excipients are selected from neutrallipids, steroids, and polymer conjugated lipids. In one embodiment, thetherapeutic agent is encapsulated within or associated with the lipidnanoparticle.

In one embodiment, provided herein is a nanoparticle composition (lipidnanoparticle) comprising:

-   -   i) between 40 and 50 mol percent of a cationic lipid;    -   ii) a neutral lipid;    -   iii) a steroid;    -   iv) a polymer conjugated lipid; and    -   v) a therapeutic agent.

As used herein, “mol percent” refers to a component's molar percentagerelative to total mols of all lipid components in the LNP (i.e., totalmols of cationic lipid(s), the neutral lipid, the steroid and thepolymer conjugated lipid).

In one embodiment, the lipid nanoparticle comprises from 41 to 49 molpercent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent,from 46 to 48 mol percent, or from 47.2 to 47.8 mol percent of thecationic lipid. In one embodiment, the lipid nanoparticle comprisesabout 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0mol percent of the cationic lipid.

In one embodiment, the neutral lipid is present in a concentrationranging from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 molpercent. In one embodiment, the neutral lipid is present in aconcentration of about 9.5, 10 or 10.5 mol percent. In one embodiment,the molar ratio of the cationic lipid to the neutral lipid ranges fromabout 4.1:1.0 to about 4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, orfrom about 4.7:1.0 to 4.8:1.0.

In one embodiment, the steroid is present in a concentration rangingfrom 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molarpercent, from 40 to 42 molar percent, from 42 to 44 molar percent, orfrom 44 to 46 molar percent. In one embodiment, the steroid is presentin a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. Inone embodiment, the molar ratio of cationic lipid to the steroid rangesfrom 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In one embodiment,the steroid is cholesterol.

In one embodiment, the therapeutic agent to lipid ratio in the LNP(i.e., N/P, were N represents the moles of cationic lipid and Prepresents the moles of phosphate present as part of the nucleic acidbackbone) range from 2:1 to 30:1, for example 3:1 to 22:1. In oneembodiment, N/P ranges from 6:1 to 20:1 or 2:1 to 12:1. Exemplary N/Pranges include about 3:1. About 6:1, about 12:1 and about 22:1.

In one embodiment, provided herein is a lipid nanoparticle comprising:

-   -   i) a cationic lipid having an effective pKa greater than        6.0; ii) from 5 to 15 mol percent of a neutral lipid;    -   iii) from 1 to 15 mol percent of an anionic lipid;    -   iv) from 30 to 45 mol percent of a steroid;    -   v) a polymer conjugated lipid; and    -   vi) a therapeutic agent, or a pharmaceutically acceptable salt        or prodrug thereof,    -   wherein the mol percent is determined based on total mol of        lipid present in the lipid nanoparticle.

In one embodiment, the cationic lipid can be any of a number of lipidspecies which carry a net positive charge at a selected pH, such asphysiological pH. Exemplary cationic lipids are described herein below.In one embodiment, the cationic lipid has a pKa greater than 6.25. Inone embodiment, the cationic lipid has a pKa greater than 6.5. In oneembodiment, the cationic lipid has a pKa greater than 6.1, greater than6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than6.45, greater than 6.55, greater than 6.6, greater than 6.65, or greaterthan 6.7.

In one embodiment, the lipid nanoparticle comprises from 40 to 45 molpercent of the cationic lipid. In one embodiment, the lipid nanoparticlecomprises from 45 to 50 mole percent of the cationic lipid.

In one embodiment, the molar ratio of the cationic lipid to the neutrallipid ranges from about 2:1 to about 8:1. In one embodiment, the lipidnanoparticle comprises from 5 to 10 mol percent of the neutral lipid.

Exemplary anionic lipids include, but are not limited to,phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG) or1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG).

In one embodiment, the lipid nanoparticle comprises from 1 to 10 molepercent of the anionic lipid. In one embodiment, the lipid nanoparticlecomprises from 1 to 5 mole percent of the anionic lipid. In oneembodiment, the lipid nanoparticle comprises from 1 to 9 mole percent,from 1 to 8 mole percent, from 1 to 7 mole percent, or from 1 to 6 molepercent of the anionic lipid. In one embodiment, the mol ratio ofanionic lipid to neutral lipid ranges from 1:1 to 1:10.

In one embodiment, the steroid cholesterol. In one embodiment, the molarratio of the cationic lipid to cholesterol ranges from about 5:1 to 1:1.In one embodiment, the lipid nanoparticle comprises from 32 to 40 molpercent of the steroid.

In one embodiment, the sum of the mol percent of neutral lipid and molpercent of anionic lipid ranges from 5 to 15 mol percent. In oneembodiment, wherein the sum of the mol percent of neutral lipid and molpercent of anionic lipid ranges from 7 to 12 mol percent.

In one embodiment, the mol ratio of anionic lipid to neutral lipidranges from 1:1 to 1:10. In one embodiment, the sum of the mol percentof neutral lipid and mol percent steroid ranges from 35 to 45 molpercent.

In one embodiment, the lipid nanoparticle comprises:

-   -   i) from 45 to 55 mol percent of the cationic lipid;    -   ii) from 5 to 10 mol percent of the neutral lipid;    -   iii) from 1 to 5 mol percent of the anionic lipid; and    -   iv) from 32 to 40 mol percent of the steroid.

In one embodiment, the lipid nanoparticle comprises from 1.0 to 2.5 molpercent of the conjugated lipid. In one embodiment, the polymerconjugated lipid is present in a concentration of about 1.5 mol percent.

In one embodiment, the neutral lipid is present in a concentrationranging from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 molpercent. In one embodiment, the neutral lipid is present in aconcentration of about 9.5, 10 or 10.5 mol percent. In one embodiment,the molar ratio of the cationic lipid to the neutral lipid ranges fromabout 4.1:1.0 to about 4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, orfrom about 4.7:1.0 to 4.8:1.0.

In one embodiment, the steroid is cholesterol. In some embodiments, thesteroid is present in a concentration ranging from 39 to 49 molarpercent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molarpercent. In one embodiment, the steroid is present in a concentration of40, 41, 42, 43, 44, 45, or 46 molar percent. In certain embodiments, themolar ratio of cationic lipid to the steroid ranges from 1.0:0.9 to1.0:1.2, or from 1.0:1.0 to 1.0:1.2.

In one embodiment, the molar ratio of cationic lipid to steroid rangesfrom 5:1 to 1:1.

In one embodiment, the lipid nanoparticle comprises from 1.0 to 2.5 molpercent of the conjugated lipid. In one embodiment, the polymerconjugated lipid is present in a concentration of about 1.5 mol percent.

In one embodiment, the molar ratio of cationic lipid to polymerconjugated lipid ranges from about 100:1 to about 20:1. In oneembodiment, the molar ratio of cationic lipid to the polymer conjugatedlipid ranges from about 35:1 to about 25:1.

In one embodiment, the molar ratio of cationic lipid to polymerconjugated lipid ranges from about 100:1 to about 20:1. In oneembodiment, the molar ratio of cationic lipid to the polymer conjugatedlipid ranges from about 35:1 to about 25:1.

In one embodiment, the lipid nanoparticle has a mean diameter rangingfrom 50 nm to 100 nm, or from 60 nm to 85 nm.

In one embodiment, the composition comprises a cationic lipid providedherein, DSPC, cholesterol, and PEG-lipid, and mRNA. In one embodiment,the a cationic lipid provided herein, DSPC, cholesterol, and PEG-lipidare at a molar ratio of about 50:10:38.5:1.5.

Nanoparticle compositions can be designed for one or more specificapplications or targets. For example, a nanoparticle composition can bedesigned to deliver a therapeutic and/or prophylactic agent such as anRNA to a particular cell, tissue, organ, or system or group thereof in amammal's body. Physiochemical properties of nanoparticle compositionscan be altered in order to increase selectivity for particular bodilytargets. For instance, particle sizes can be adjusted based on thefenestration sizes of different organs. The therapeutic and/orprophylactic agent included in a nanoparticle composition can also beselected based on the desired delivery target or targets. For example, atherapeutic and/or prophylactic agent can be selected for a particularindication, condition, disease, or disorder and/or for delivery to aparticular cell, tissue, organ, or system or group thereof (e.g.,localized or specific delivery). In certain embodiments, a nanoparticlecomposition can include an mRNA encoding a polypeptide of interestcapable of being translated within a cell to produce the polypeptide ofinterest. Such a composition can be designed to be specificallydelivered to a particular organ. In certain embodiments, a compositioncan be designed to be specifically delivered to a mammalian liver.

The amount of a therapeutic and/or prophylactic agent in a nanoparticlecomposition can depend on the size, composition, desired target and/orapplication, or other properties of the nanoparticle composition as wellas on the properties of the therapeutic and/or prophylactic agent. Forexample, the amount of an RNA useful in a nanoparticle composition candepend on the size, sequence, and other characteristics of the RNA. Therelative amounts of a therapeutic and/or prophylactic agent and otherelements (e.g., lipids) in a nanoparticle composition can also vary. Insome embodiments, the wt/wt ratio of the lipid component to atherapeutic and/or prophylactic agent in a nanoparticle composition canbe from about 5:1 to about 60:1, such as about 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 22:1,25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt/wtratio of the lipid component to a therapeutic and/or prophylactic agentcan be from about 10:1 to about 40:1. In certain embodiments, the wt/wtratio is about 20:1. The amount of a therapeutic and/or prophylacticagent in a nanoparticle composition can, for example, be measured usingabsorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

In some embodiments, a nanoparticle composition includes one or moreRNAs, and the one or more RNAs, lipids, and amounts thereof can beselected to provide a specific N:P ratio. The N:P ratio of thecomposition refers to the molar ratio of nitrogen atoms in one or morelipids to the number of phosphate groups in an RNA. In some embodiments,a lower N:P ratio is selected. The one or more RNA, lipids, and amountsthereof can be selected to provide an N:P ratio from about 2:1 to about30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1,16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certainembodiments, the N:P ratio can be from about 2:1 to about 8:1. In otherembodiments, the N:P ratio is from about 5:1 to about 8:1. For example,the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio may beabout 5.67:1.

The physical properties of a nanoparticle composition can depend on thecomponents thereof. For example, a nanoparticle composition includingcholesterol as a structural lipid can have different characteristicscompared to a nanoparticle composition that includes a differentstructural lipid. Similarly, the characteristics of a nanoparticlecomposition can depend on the absolute or relative amounts of itscomponents. For instance, a nanoparticle composition including a highermolar fraction of a phospholipid may have different characteristics thana nanoparticle composition including a lower molar fraction of aphospholipid. Characteristics may also vary depending on the method andconditions of preparation of the nanoparticle composition.

Nanoparticle compositions may be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) may be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) may beused to measure zeta potentials. Dynamic light scattering may also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential.

In various embodiments, the mean size of a nanoparticle composition canbe between 10s of nm and 100s of nm. For example, the mean size can befrom about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or150 nm. In some embodiments, the mean size of a nanoparticle compositioncan be from about 50 nm to about 100 nm, from about 50 nm to about 90nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm,from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, fromabout 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nmto about 90 nm, from about 70 nm to about 80 nm, from about 80 nm toabout 100 nm, from about 80 nm to about 90 nm, or from about 90 nm toabout 100 nm. In certain embodiments, the mean size of a nanoparticlecomposition can be from about 70 nm to about 100 nm. In someembodiments, the mean size can be about 80 nm. In other embodiments, themean size can be about 100 nm.

A nanoparticle composition can be relatively homogenous. Apolydispersity index can be used to indicate the homogeneity of ananoparticle composition, e.g., the particle size distribution of thenanoparticle compositions. A small (e.g., less than 0.3) polydispersityindex generally indicates a narrow particle size distribution. Ananoparticle composition can have a polydispersity index from about 0 toabout 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersityindex of a nanoparticle composition can be from about 0.10 to about0.20.

The zeta potential of a nanoparticle composition can be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential can describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species caninteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition can be from about −10 mV to about +20 mV, from about −10 mVto about +15 mV, from about −10 mV to about +10 mV, from about −10 mV toabout +5 mV, from about −10 mV to about 0 mV, from about −10 mV to about−5 mV, from about −5 mV to about +20 mV, from about −5 mV to about +15mV, from about −5 mV to about +10 mV, from about −5 mV to about +5 mV,from about −5 mV to about 0 mV, from about 0 mV to about +20 mV, fromabout 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mVto about +15 mV, or from about +5 mV to about +10 mV.

The efficiency of encapsulation of a therapeutic and/or prophylacticagent describes the amount of therapeutic and/or prophylactic agent thatis encapsulated or otherwise associated with a nanoparticle compositionafter preparation, relative to the initial amount provided. Theencapsulation efficiency is desirably high (e.g., close to 100%). Theencapsulation efficiency can be measured, for example, by comparing theamount of therapeutic and/or prophylactic agent in a solution containingthe nanoparticle composition before and after breaking up thenanoparticle composition with one or more organic solvents ordetergents. Fluorescence can be used to measure the amount of freetherapeutic and/or prophylactic agent (e.g., RNA) in a solution. For thenanoparticle compositions described herein, the encapsulation efficiencyof a therapeutic and/or prophylactic agent can be at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulationefficiency can be at least 80%. In certain embodiments, theencapsulation efficiency can be at least 90%.

A nanoparticle composition can optionally comprise one or more coatings.For example, a nanoparticle composition can be formulated in a capsule,film, or tablet having a coating. A capsule, film, or tablet including acomposition described herein can have any useful size, tensile strength,hardness, or density.

6.4.7 Pharmaceutical Compositions

According to the present disclosure, nanoparticle compositions can beformulated in whole or in part as pharmaceutical compositions.Pharmaceutical compositions can include one or more nanoparticlecompositions. For example, a pharmaceutical composition can include oneor more nanoparticle compositions including one or more differenttherapeutic and/or prophylactic agents. Pharmaceutical compositions canfurther include one or more pharmaceutically acceptable excipients oraccessory ingredients such as those described herein. General guidelinesfor the formulation and manufacture of pharmaceutical compositions andagents are available, for example, in Remington's The Science andPractice of Pharmacy, 21′ Edition, A. R. Gennaro; Lippincott, Williams &Wilkins, Baltimore, Md., 2006. Conventional excipients and accessoryingredients can be used in any pharmaceutical composition, exceptinsofar as any conventional excipient or accessory ingredient can beincompatible with one or more components of a nanoparticle composition.An excipient or accessory ingredient can be incompatible with acomponent of a nanoparticle composition if its combination with thecomponent can result in any undesirable biological effect or otherwisedeleterious effect.

In some embodiments, one or more excipients or accessory ingredients canmake up greater than 50% of the total mass or volume of a pharmaceuticalcomposition including a nanoparticle composition. For example, the oneor more excipients or accessory ingredients can make up 50%, 60%, 70%,80%, 90%, or more of a pharmaceutical convention. In some embodiments, apharmaceutically acceptable excipient is at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% pure. In someembodiments, an excipient is approved for use in humans and forveterinary use. In some embodiments, an excipient is approved by UnitedStates Food and Drug Administration. In some embodiments, an excipientis pharmaceutical grade. In some embodiments, an excipient meets thestandards of the United States Pharmacopoeia (USP), the EuropeanPharmacopoeia (EP), the British Pharmacopoeia, and/or the InternationalPharmacopoeia.

Relative amounts of the one or more nanoparticle compositions, the oneor more pharmaceutically acceptable excipients, and/or any additionalingredients in a pharmaceutical composition in accordance with thepresent disclosure will vary, depending upon the identity, size, and/orcondition of the subject treated and further depending upon the route bywhich the composition is to be administered. By way of example, apharmaceutical composition can comprise between 0.1% and 100% (wt/wt) ofone or more nanoparticle compositions.

In certain embodiments, the nanoparticle compositions and/orpharmaceutical compositions of the disclosure are refrigerated or frozenfor storage and/or shipment (e.g., being stored at a temperature of 4°C. or lower, such as a temperature between about −150° C. and about 0°C. or between about −80° C. and about −20° C. (e.g., about −5° C., −10°C., −15° C., −20° C., −25° C., 30° C., −40° C., −50° C., −60° C., −70°C., −80° C., −90° C., −130° C. or −150° C.). For example, thepharmaceutical composition comprising a compound of any of Formulae (I)to (IV) (and sub-formulas thereof) is a solution that is refrigeratedfor storage and/or shipment at, for example, about −20° C., −30° C.,−40° C., −50° C., −60° C., −70° C., or −80° C. In certain embodiments,the disclosure also relates to a method of increasing stability of thenanoparticle compositions and/or pharmaceutical compositions comprisinga compound of any of Formulae (I) to (IV) (and sub-formulas thereof) bystoring the nanoparticle compositions and/or pharmaceutical compositionsat a temperature of 4° C. or lower, such as a temperature between about−150° C. and about 0° C. or between about −80° C. and about −20° C.,e.g., about −5° C., −10° C., −15° C., −20° C., −25° C., −30° C., 40° C.,−50° C., −60° C., −70° C., −80° C., −90° C., −130° C. or −150° C.). Forexample, the nanoparticle compositions and/or pharmaceuticalcompositions disclosed herein are stable for about at least 1 week, atleast 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, atleast 6 weeks, at least 1 month, at least 2 months, at least 4 months,at least 6 months, at least 8 months, at least 10 months, at least 12months, at least 14 months, at least 16 months, at least 18 months, atleast 20 months, at least 22 months, or at least 24 months, e.g., at atemperature of 4° C. or lower (e.g., between about 4° C. and −20° C.).In one embodiment, the formulation is stabilized for at least 4 weeks atabout 4° C. In certain embodiments, the pharmaceutical composition ofthe disclosure comprises a nanoparticle composition disclosed herein anda pharmaceutically acceptable carrier selected from one or more of Tris,an acetate (e.g., sodium acetate), an citrate (e.g., sodium citrate),saline, PBS, and sucrose. In certain embodiments, the pharmaceuticalcomposition of the disclosure has a pH value between about 7 and 8(e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or8.0, or between 7.5 and 8 or between 7 and 7.8). For example, apharmaceutical composition of the disclosure comprises a nanoparticlecomposition disclosed herein, Tris, saline and sucrose, and has a pH ofabout 7.5-8, which is suitable for storage and/or shipment at, forexample, about −20° C. For example, a pharmaceutical composition of thedisclosure comprises a nanoparticle composition disclosed herein and PBSand has a pH of about 7-7.8, suitable for storage and/or shipment at,for example, about 4° C. or lower. “Stability,” “stabilized,” and“stable” in the context of the present disclosure refers to theresistance of nanoparticle compositions and/or pharmaceuticalcompositions disclosed herein to chemical or physical changes (e.g.,degradation, particle size change, aggregation, change in encapsulation,etc.) under given manufacturing, preparation, transportation, storageand/or in-use conditions, e.g., when stress is applied such as shearforce, freeze/thaw stress, etc.

Nanoparticle compositions and/or pharmaceutical compositions includingone or more nanoparticle compositions can be administered to any patientor subject, including those patients or subjects that can benefit from atherapeutic effect provided by the delivery of a therapeutic and/orprophylactic agent to one or more particular cells, tissues, organs, orsystems or groups thereof, such as the renal system. Although thedescriptions provided herein of nanoparticle compositions andpharmaceutical compositions including nanoparticle compositions areprincipally directed to compositions which are suitable foradministration to humans, it will be understood by the skilled artisanthat such compositions are generally suitable for administration to anyother mammal. Modification of compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and/or perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the compositions is contemplated include, butare not limited to, humans, other primates, and other mammals, includingcommercially relevant mammals such as cattle, pigs, hoses, sheep, cats,dogs, mice, and/or rats.

A pharmaceutical composition including one or more nanoparticlecompositions can be prepared by any method known or hereafter developedin the art of pharmacology. In general, such preparatory methods includebringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if desirable ornecessary, dividing, shaping, and/or packaging the product into adesired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosurecan be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” is discrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient (e.g., nanoparticlecomposition). The amount of the active ingredient is generally equal tothe dosage of the active ingredient which would be administered to asubject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

Pharmaceutical compositions can be prepared in a variety of formssuitable for a variety of routes and methods of administration. Forexample, pharmaceutical compositions can be prepared in liquid dosageforms (e.g., emulsions, microemulsions, nanoemulsions, solutions,suspensions, syrups, and elixirs), injectable forms, solid dosage forms(e.g., capsules, tablets, pills, powders, and granules), dosage formsfor topical and/or transdermal administration (e.g., ointments, pastes,creams, lotions, gels, powders, solutions, sprays, inhalants, andpatches), suspensions, powders, and other forms.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, nanoemulsions, solutions, suspensions, syrups, and/orelixirs. In addition to active ingredients, liquid dosage forms cancomprise inert diluents commonly used in the art such as, for example,water or other solvents, solubilizing agents and emulsifiers such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadditional therapeutic and/or prophylactic agents, additional agentssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and/or perfuming agents. In certain embodiments forparenteral administration, compositions are mixed with solubilizingagents such as Cremophor™, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations can be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

The disclosure features methods of delivering a therapeutic and/orprophylactic agent to a mammalian cell or organ, producing a polypeptideof interest in a mammalian cell, and treating a disease or disorder in amammal in need thereof comprising administering to a mammal and/orcontacting a mammalian cell with a nanoparticle composition including atherapeutic and/or prophylactic agent.

6.5 Methods

In one aspect, provided herein are also methods for managing, preventingand treating an infectious disease caused by coronavirus infection in asubject. In some embodiments, the infectious disease being managed,prevented or treated with the methods described herein is caused byinfection of a coronavirus selected from SARS-CoV-2, severe acuterespiratory syndrome coronavirus (SARS-CoV), Middle East respiratorysyndrome coronavirus (MERS-CoV), human coronavirus NL63 (HCoV-NL63),human coronavirus 0C₄₃, the porcine epidemic diarrhea coronavirus(PEDV), porcine transmissible gastroenteritis coronavirus (TGEV),porcine respiratory coronavirus (PRCV), bat coronavirus HKU4, mousehepatitis coronavirus (MHV), bovine coronavirus (BCoV), avian infectiousbronchitis coronavirus (IBV), porcine deltacoronavirus (PdCV).

In specific embodiments, the infectious disease being managed, preventedor treated with the methods described herein is caused by coronavirusinfection of the respiratory system, the nervous system, the immunesystem, the digestion system and/or a major organ of the subject (e.g.,a human or non-human mammal). In specific embodiments, the infectiousdisease being managed, prevented or treated with the methods describedherein is respiratory tract infection, lung infection, renal infection,liver infection, enteric infection, neurologic infections, respiratorysyndrome, bronchitis, pneumonia, gastroenteritis, encephalomyelitis,encephalitis, sarcoidosis, diarrhea, hepatitis, or demyelinatingdisease. In specific embodiments, the infectious disease is respiratorytract infection, lung infection, pneumonia or respiratory syndromecaused by infection by the SARS-CoV-2.

In some embodiments, the present method for managing, preventing andtreating an infectious disease caused by coronavirus infection in asubject comprises administering to the subject a therapeutic effectiveamount of a therapeutic nucleic acid as described herein. In specificembodiments, the therapeutic nucleic acid is an mRNA molecule asdescribed herein.

In some embodiments, the present method for managing, preventing andtreating an infectious disease caused by coronavirus infection in asubject comprises administering to the subject a therapeutic effectiveamount of a therapeutic composition comprising a therapeutic nucleicacid as described herein. In specific embodiments, the therapeuticnucleic acid is an mRNA molecule as described herein.

In some embodiments, the present method for managing, preventing andtreating an infectious disease caused by coronavirus infection in asubject comprises administering to the subject a therapeutic effectiveamount of a vaccine composition comprising a therapeutic nucleic acid asdescribed herein. In specific embodiments, the therapeutic nucleic acidis an mRNA molecule as described herein.

In some embodiments, the present method for managing, preventing andtreating an infectious disease caused by coronavirus infection in asubject comprises administering to the subject a therapeutic effectiveamount of a lipid-containing composition comprising a therapeuticnucleic acid as described herein. In specific embodiments, thetherapeutic nucleic acid is an mRNA molecule as described herein.

In some embodiments, the present method for managing, preventing andtreating an infectious disease caused by coronavirus infection in asubject comprises administering to the subject a therapeutic effectiveamount of a lipid-containing composition comprising a therapeuticnucleic acid as described herein, wherein the lipid-containingcomposition is formulated as a lipid nanoparticle encapsulating thetherapeutic nucleic acid in a lipid shell. In specific embodiments, thetherapeutic nucleic acid is an mRNA molecule as described herein. Inspecific embodiments, the cells in the subject effectively intake thelipid-containing composition (e.g., lipid nanoparticles) describedherein upon administration. In specific embodiments, lipid-containingcomposition (e.g., lipid nanoparticles) described herein are endocytosedby cells of the subject.

In some embodiments, upon administration to a subject in need thereof ofthe therapeutic nucleic acid as described herein, a vaccine compositioncomprising the therapeutic nucleic acids described herein, alipid-containing composition (e.g., lipid nanoparticles) comprising thetherapeutic nucleic acids described herein, the cells in the subjectuptake and express the administered therapeutic nucleic acids to producea peptide or polypeptide encoded by the nucleic acid. In someembodiments, the encoded peptide or polypeptide is derived from thecoronavirus causing the infectious disease being managed, prevented, ortreated by the method.

6.5.1 Immune Responses

In some embodiments, upon administration to a subject in need thereof ofthe therapeutic nucleic acid as described herein, a vaccine compositioncomprising the therapeutic nucleic acids described herein, alipid-containing composition (e.g., lipid nanoparticles) comprising thetherapeutic nucleic acids described herein, one or more immune responsesagainst the coronavirus is elicited in the subject. In some embodiments,the elicited immune response comprises one or more adaptive immuneresponses against the coronavirus. In some embodiments, the elicitedimmune response comprises one or more innate immune responses againstthe coronavirus. The one or more immune responses can be in the form of,e.g., an antibody response (humoral response) or a cellular immuneresponse, e.g., cytokine secretion (e.g., interferon-gamma), helperactivity or cellular cytotoxicity. In some embodiments, expression of anactivation marker on immune cells, expression of a co-stimulatoryreceptor on immune cells, expression of a ligand for a co-stimulatoryreceptor, cytokine secretion, infiltration of immune cells (e.g.,T-lymphocytes, B lymphocytes and/or NK cells) to a infected cell,production of antibody specifically recognizing one or more viralproteins (e.g., the viral peptide or protein encoded by the therapeuticnucleic acid), effector function, T cell activation, T celldifferentiation, T cell proliferation, B cell differentiation, B cellproliferation, and/or NK cell proliferation is induced, activated and/orenhanced. In some embodiments, activation and proliferation ofmyeloid-derived suppressor cell (MDSC) and Treg cells are inhibited.

In some embodiments, upon administration to a subject in need thereof ofthe therapeutic nucleic acid as described herein, a vaccine compositioncomprising the therapeutic nucleic acids described herein, alipid-containing composition (e.g., lipid nanoparticles) comprising thetherapeutic nucleic acids described herein, one or more neutralizingantibody against the coronavirus or cells infected by the coronavirus isproduced in the subject.

In specific embodiments, the neutralizing antibody specifically binds toone or more epitopes of the S protein of the coronavirus and inhibits orreduces one or more S protein function or activity. In specificembodiments, binding of the S protein to its cellular receptor isreduced or inhibited. In specific embodiments, binding of thecoronavirus S protein to angiotensin-converting enzyme 2 (ACE2),aminopeptidase N (APN), dipeptidyl peptidase 4 (DPP4), carcinoembryonicantigen-related cell adhesion molecule 1 (CEACAM1) and/or sugar on thehost cell surface is reduced or inhibited. In specific embodiments,attachment of the coronavirus with host cells in the subject is reducedor inhibited. In specific embodiments, host cell membrane fusion inducedby the coronavirus is reduced or inhibited. In specific embodiments,infection (e.g., entry) of host cells in the subject by the coronavirusis reduced or inhibited. In some embodiments, the neutralizing antibodyreduces the S protein function or activity by 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100%.

In another embodiments, the neutralizing antibody against thecoronavirus or cells infected by the coronavirus is produced in thesubject. In specific embodiments, the neutralizing antibody specificallybinds to one or more epitopes of the N protein of the coronavirus, andinhibits or reduces one or more N protein function or activity. Inspecific embodiments, binding of the coronavirus N protein to reproducedviral genomic sequences is reduced or inhibited. In specificembodiments, packaging of reproduced viral genomic sequence into afunctional viral capsid is reduced or inhibited. In specificembodiments, reproduction of viable progenies of the coronavirus isreduced or inhibited. In some embodiments, the neutralizing antibodyreduces the S protein function or activity by 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100%.

In specific embodiments, the neutralizing antibody binds to one or moreviral proteins present on a viral particle or the surface of infectedcells, and mark the viral particles or infected cells for destruction bythe subject's immune system. In some embodiments, endocytosis of viralparticles by white blood cells (e.g., macrophage) is induced orenhanced. In some embodiments, antibody-dependent cell-mediatedcytotoxicity (ADCC) against infected cells in the subject is induced orenhanced. In some embodiments, antibody-dependent cellular phagocytosis(ADCP) against infected cells in the subject is induced or enhanced. Insome embodiments, complement dependent cytotoxicity (CDC) againstinfected cells in the subject is induced or enhanced.

6.5.2 Combination Therapy

In some embodiments, the composition of the present disclosure canfurther comprise one or more additional therapeutic agents. In someembodiments, the additional therapeutic agent is an adjuvant capable ofbolstering immunogenicity of the composition (e.g., a genetic vaccine).In some embodiments, the additional therapeutic agent is an immunemodulator that enhances immune responses in a subject. In someembodiments, the adjuvant and the therapeutic nucleic acid in thecomposition can have a synergistic action in eliciting an immuneresponse in a subject.

In some embodiments, the additional therapeutic agent and thetherapeutic nucleic acid of the present disclosure can be co-formulatedin one composition. For example, the additional therapeutic agent can beformulated as part of the composition comprising the therapeutic nucleicacid of the present disclosure. Alternatively, in some embodiments, theadditional therapeutic agent and therapeutic nucleic acid of the presentdisclosure can be formulated as separate compositions or dose units forco-administration either sequentially or simultaneously to a subject.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure is formulated as part of a lipid-containing composition asdescribed in Section 6.4, and the additional therapeutic agent isformulated as a separate composition. In particular embodiments, thetherapeutic nucleic acid of the present disclosure is formulated as partof a lipid-containing composition as described in Section 6.4, whereinthe additional therapeutic agent is also formulated as part of thelipid-containing composition.

In particular embodiments, the therapeutic nucleic acid of the presentdisclosure is formulated so that the therapeutic nucleic acid isencapsulated in a lipid shell of a lipid nanoparticle as described inSection 6.4, and the additional therapeutic agent is formulated as aseparate composition. In particular embodiments, the therapeutic nucleicacid of the present disclosure is formulated so that the therapeuticnucleic acid is encapsulated in a lipid shell of a lipid nanoparticle asdescribed in Section 6.4, wherein the lipid nanoparticles also enclosethe additional therapeutic agent molecule or a nucleic acid encoding theadditional therapeutic agent molecule. In particular embodiments, thetherapeutic nucleic acid of the present disclosure is formulated so thatthe therapeutic nucleic acid is encapsulated in a lipid shell of a lipidnanoparticle as described in Section 6.4, wherein the lipidnanoparticles and the additional therapeutic agent are formulated into asingle composition.

In specific embodiments, the additional therapeutic agent is anadjuvant. In some embodiments, the adjuvant comprises an agent thatpromotes maturation of dendritic cells (DCs) in a vaccinated subject,such as but not limited to lipopolysaccharides, TNF-alpha or CD40ligand. In some embodiments, the adjuvant is an agent that recognized bythe immune system of the vaccinated subject as a “danger signal,” suchas LPS, GP96, etc.

In some embodiments, the adjuvant comprises an immunostimulatingcytokine such as but not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta,INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors,such as hGH.

In some embodiments, the adjuvant comprises a compound known as capableof eliciting an innate immune response. One exemplary class of suchcompound are Toll-like receptor ligands, such as ligands of humanToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, and ligands of murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.Another exemplar class of such compounds are immuno-stimulating nucleicacids, such as oligonucleotides containing the CpG motif. CpG containingnucleic acids can be DNA (CpG-DNA) or RNA (CpG-RNA) molecules. A CpG-RNAor CpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), adouble-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA)or a double-stranded CpG-RNA (ds CpG-RNA). In some embodiments, the CpGnucleic acid is in the form of CpG-RNA. In particular embodiments, theCpG nucleic acid is in the form of single-stranded CpG-RNA (ss CpG-RNA).In some embodiments, the CpG nucleic acid contains at least one or more(mitogenic) cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). Insome embodiments, at least one CpG motif contained in these sequences(i.e., the C (cytosine) and/or the G (guanine) forming the CpG motif) isunmethylated.

In some embodiments, the additional therapeutic agent is an immunemodulator that activate, boost or restore normal immune functions. Inspecific embodiments, the immune modulator is an agonist of aco-stimulatory signal of an immune cell, such as a T-lymphocyte, NK cellor antigen-presenting cell (e.g., a dendritic cell or macrophage). Inspecific embodiments, the immune modulator is an antagonist of aninhibitory signal of an immune cell, such as a T-lymphocyte, NK cell orantigen-presenting cell (e.g., a dendritic cell or macrophage).

Various immune cell stimulatory agents are known to one of skill in theart and can be used in connection with the present disclosure. Incertain embodiments, the agonist of a co-stimulatory signal is anagonist of a co-stimulatory molecule (e.g., co-stimulatory receptor)found on immune cells, such as, T-lymphocytes (e.g., CD4+ orCD8+T-lymphocytes), NK cells and/or antigen-presenting cells (e.g.,dendritic cells or macrophages). Specific examples of co-stimulatorymolecules include glucocorticoid-induced tumor necrosis factor receptor(GITR), Inducible T-cell costimulator (ICOS or CD278), OX40 (CD134),CD27, CD28, 4-IBB (CD137), CD40, lymphotoxin alpha (LT alpha), LIGHT(lymphotoxin-like, exhibits inducible expression, and competes withherpes simplex virus glycoprotein D for HVEM, a receptor expressed by Tlymphocytes), CD226, cytotoxic and regulatory T cell molecule (CRT AM),death receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembraneactivator and CAML interactor (TALI), B cell-activating factor receptor(BAFFR), and B cell maturation protein (BCMA).

In specific embodiments, the agonist of a co-stimulatory receptor is anantibody or antigen-binding fragment thereof that specifically binds tothe co-stimulatory receptor. Specific examples of co-stimulatoryreceptors include GITR, ICOS, OX40, CD27, CD28, 4-1BB, CD40, LT alpha,LIGHT, CD226, CRT AM, DR3, LTBR, TALI, BAFFR, and BCMA. In certainspecific embodiments, the antibody is a monoclonal antibody. In otherspecific embodiments, the antibody is an sc-Fv. In a specificembodiment, the antibody is a bispecific antibody that binds to tworeceptors on an immune cell. In other embodiments, the bispecificantibody binds to a receptor on an immune cell and to another receptoron a virus infected diseased cell. In specific embodiments, the antibodyis a human or humanized antibody.

In another embodiment, the agonist of a co-stimulatory receptor is aligand of the co-stimulatory receptor or a functional derivativethereof. In certain embodiments, the ligand is fragment of a nativeligand. Specific examples of native ligands include ICOSL, B7RP1,CD137L, OX40L, CD70, herpes virus entry mediator (HVEM), CD80, and CD86.The nucleotide sequences encoding native ligands as well as the aminoacid sequences of native ligands are known in the art.

In specific embodiments, the antagonist is an antagonist of aninhibitory molecule (e.g., inhibitory receptor) found on immune cells,such as, e.g., T-lymphocytes (e.g., CD4+ or CD8+ T-lymphocytes), NKcells and/or antigen-presenting cells (e.g., dendritic cells ormacrophages). Specific examples of inhibitory molecules includecytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD52), programmedcell death protein 1 (PD1 or CD279), B and T-lymphocyte attenuator(BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyteactivation gene 3 (LAG3), T-cell membrane protein 3 (TIM3), CD 160,adenosine A2a receptor (A2aR), T cell immunoreceptor with immunoglobulinand ITIM domains (TIGIT), leukocyte-associated immunoglobulin-likereceptor 1 (LAIR1), and CD 160.

In another embodiment, the antagonist of an inhibitory receptor is anantibody (or an antigen-binding fragment) that specifically binds to thenative ligand for the inhibitory receptor and blocks the native ligandfrom binding to the inhibitory receptor and transducing an inhibitorysignal(s). In certain specific embodiments, the antibody is a monoclonalantibody. In other specific embodiments, the antibody is an sc-Fv. In aspecific embodiment, the antibody is a bispecific antibody that binds totwo receptors on an immune cell. In other embodiments, the bispecificantibody binds to a receptor on an immune cell and to another receptoron a virus infected diseased cell. In specific embodiments, the antibodyis a human or humanized antibody.

In another embodiments, the antagonist of an inhibitory receptor is asoluble receptor or a functional derivative thereof that specificallybinds to the native ligand for the inhibitory receptor and blocks thenative ligand from binding to the inhibitory receptor and transducing aninhibitory signal(s). Specific examples of native ligands for inhibitoryreceptors include PDL-1, PDL-2, B7-H3, B7-H4, HVEM, Ga19 and adenosine.Specific examples of inhibitory receptors that bind to a native ligandinclude CTLA-4, PD-1, BTLA, KIR, LAG3, TIM3, and A2aR.

In another embodiment, the antagonist of an inhibitory receptor is anantibody (or an antigen-binding fragment) or ligand that binds to theinhibitory receptor, but does not transduce an inhibitory signal(s).Specific examples of inhibitory receptors include CTLA-4, PD1, BTLA,KIR, LAG3, TIM3, and A2aR. In certain specific embodiments, the antibodyis a monoclonal antibody. In other specific embodiments, the antibody isan scFv. In particular embodiments, the antibody is a human or humanizedantibody. A specific example of an antibody to inhibitory receptor isanti-CTLA-4 antibody (Leach D R, et al. Science 1996; 271: 1734-1736).Another example of an antibody to inhibitory receptor is anti-PD-1antibody (Topalian SL, NEJM 2012; 28:3167-75).

6.5.3 Patient Population

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy described herein is administered to a subject inneed thereof.

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy described herein is administered to a human subject.In some embodiments, a subject administered with a therapeutic nucleicacid described herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising a therapeutic nucleic acids describedherein or a combination therapy described herein is an elderly human. Insome embodiments, a subject administered with a therapeutic nucleic aciddescribed herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein or a combination therapy described herein is a human adult. Insome embodiments, a subject administered with a therapeutic nucleic aciddescribed herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein or a combination therapy described herein is human child. In someembodiments, a subject administered with a therapeutic nucleic aciddescribed herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein or a combination therapy described herein is human toddler. Insome embodiments, a subject administered with a therapeutic nucleic aciddescribed herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein or a combination therapy described herein is human infant.

In some embodiments, a subject administered with a therapeutic nucleicacid described herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein or a combination therapy described herein is administered to anon-human mammal.

In some embodiments, a subject administered with a therapeutic nucleicacid described herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein, or the combination therapy described herein is administered to asubject exhibiting at least one symptom associated with coronavirusinfection. In some embodiments, the subject receiving administration ofa therapeutic nucleic acid described herein, a vaccine compositioncomprising the therapeutic nucleic acids described herein, alipid-containing composition (e.g., lipid nanoparticles) comprising thetherapeutic nucleic acids described herein, or a combination therapydescribed herein exhibits one or more symptoms of upper respiratorytract infection, lower respiratory tract infection, lung infection,renal infection, liver infection, enteric infection, hepatic infection,neurologic infections, respiratory syndrome, pneumonia, gastroenteritis,encephalomyelitis, encephalitis, sarcoidosis, diarrhea, hepatitis, anddemyelinating disease.

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy as described herein is administered to a subjectthat is asymptomatic for coronavirus infection.

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy described herein is administered to a subject who isat risk of, or susceptible to, coronavirus infection. In someembodiments, a subject at risk of, or susceptible to, coronavirusinfection is an elderly human. In some embodiments, a subject at riskof, or susceptible to, coronavirus infection is a human adult. In someembodiments, a subject at risk of, or susceptible to, coronavirusinfection is a human child. In some embodiments, a subject at risk of,or susceptible to, coronavirus infection is a human adult toddler. Insome embodiments, a subject at risk of, or susceptible to, coronavirusinfection is a human adult infant. In some embodiments, a subject atrisk of, or susceptible to, coronavirus infection is a human subjecthaving existing health condition that affects the subject's immunesystem. In some embodiments, a subject at risk of, or susceptible to,coronavirus infection is a human subject having existing healthcondition that affects the subject's major organs. In some embodiments,a subject at risk of, or susceptible to, coronavirus infection is ahuman subject having existing health condition that affects thesubject's lung function. In some embodiments, a subject at risk of, orsusceptible to, coronavirus infection is an elderly human subject havingan existing health condition that affects the subject's immune system,or a major organ, such as lung function. In various embodimentsdescribed in this paragraph, a subject at risk of, or susceptible to,coronavirus infection can be either exhibiting symptoms of coronavirusinfection or asymptomatic for coronavirus infection.

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy described herein is administered to a subject whohas been diagnosed positive for the coronavirus infection. In someembodiments, the subject diagnosed positive for coronavirus infection isasymptomatic for coronavirus infection, and the diagnosis is based ondetecting the presence of a viral nucleic acid or protein from a sampletaken from the subject. In some embodiments, the diagnosis is based onclinical symptoms exhibited by the patient. Exemplary symptoms that mayserve as the basis of diagnosis include but are not limited to upperrespiratory tract infection, lower respiratory tract infection, lunginfection, renal infection, liver infection, enteric infection, hepaticinfection, neurologic infections, respiratory syndrome, pneumonia,gastroenteritis, encephalomyelitis, encephalitis, sarcoidosis, diarrhea,hepatitis, and demyelinating disease. In some embodiments, the diagnosisis based on a subject's exhibited clinical symptom combined with thesubject's history of being in contact with a geographical location,population, and/or individual considered of having a high risk ofcarrying the coronavirus, such as another individual diagnosed positivefor the coronavirus infection.

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy described herein is administered to a subject whohas not previously received administration of the therapeutic nucleicacid, the vaccine composition, the lipid-containing composition (e.g.,lipid nanoparticles), or the combination therapy.

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy described herein is administered to a subject whohas previously received administration of the therapeutic nucleic acid,the vaccine composition, the lipid-containing composition (e.g., lipidnanoparticles), or the combination therapy. In specific embodiments, thesubject has been previously administered a therapeutic nucleic aciddescribed herein, the vaccine composition comprising the therapeuticnucleic acids described herein, the lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein, or the combination therapy as described herein once, twice,three times or more.

In some embodiments, a therapeutic nucleic acid described herein, avaccine composition comprising the therapeutic nucleic acids describedherein, a lipid-containing composition (e.g., lipid nanoparticles)comprising the therapeutic nucleic acids described herein, or acombination therapy described herein is administered to a subject whohas received a therapy prior to administration of the therapeuticnucleic acid, the vaccine composition, the lipid-containing composition(e.g., lipid nanoparticles), or the combination therapy. In someembodiments, the subject administered with a therapeutic nucleic aciddescribed herein, a vaccine composition comprising the therapeuticnucleic acids described herein, a lipid-containing composition (e.g.,lipid nanoparticles) comprising the therapeutic nucleic acids describedherein, or a combination therapy described herein experienced adverseside effects to a prior therapy or a prior therapy was discontinued dueto unacceptable levels of toxicity to the subject.

6.5.4 Administration Dosage and Frequency

The amount of therapeutic nucleic acid or a composition thereof whichwill be effective in the management, prevention and/or treatment ofinfectious disease will depend on the nature of the disease beingtreated, the route of administration, the general health of the subject,etc. and should be decided according to the judgment of a medicalpractitioner. Standard clinical techniques, such as in vitro assays, mayoptionally be employed to help identify optimal dosage ranges.Nevertheless, suitable dosage ranges of a therapeutic nucleic acid asdescribed herein for administration are generally about 0.001 mg, 0.005mg, 0.01 mg, 0.05 mg. 0.1 mg. 0.5 mg, 1.0 mg, 2.0 mg. 3.0 mg, 4.0 mg,5.0 mg, 10.0 mg, 0.001 mg to 10.0 mg, 0.01 mg to 1.0 mg, 0.1 mg to 1 mg,and 0.1 mg to 5.0 mg. The therapeutic nucleic acid or a compositionthereof can be administered to a subject once, twice, three, four ormore times with intervals as often as needed. Effective doses may beextrapolated from dose response curves derived from in vitro or animalmodel test systems.

In certain embodiments, a therapeutic nucleic acid or a compositionthereof is administered to a subject as a single dose followed by asecond dose 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, 1 to2 weeks later. In accordance with these embodiments, boosterinoculations may be administered to the subject at 6 to 12 monthintervals following the second inoculation.

In certain embodiments, administration of a therapeutic nucleic acid ora composition thereof may be repeated and the administrations may beseparated by at least 1 day, 2 days, 3 days, 5 days, 6 says, 7 days, 10days, 14 days, 15 days, 21 days, 28 days, 30 days, 45 days, 2 months, 75days, 3 months, or at least 6 months. In other embodiments,administration of therapeutic nucleic acid or a composition thereof maybe repeated and the administrations may be separated by 1 to 14 days, 1to 7 days, 7 to 14 days, 1 to 30 days, 15 to 30 days, 15 to 45 days, 15to 75 days, 15 to 90 days, 1 to 3 months, 3 to 6 months, 3 to 12 months,or 6 to 12 months. In some embodiments, a first therapeutic nucleic acidor a composition thereof is administered to a subject followed by theadministration of a second therapeutic nucleic acid or a compositionthereof. In certain embodiments, the first and second therapeuticnucleic acids or compositions thereof may be separated by at least 1day, 2 days, 3 days, 5 days, 6 days, 7 days, 10 days, 14 days, 15 days,21 days, 28 days, 30 days, 45 days, 2 months, 75 days, 3 months, or atleast 6 months. In other embodiments, the first and second therapeuticnucleic acids or compositions thereof may be separated by 1 to 14 days,1 to 7 days, 7 to 14 days, 1 to 30 days, 15 to 30 days, 15 to 45 days,15 to 75 days, 15 to 90 days, 1 to 3 months, 3 to 6 months, 3 to 12months, or 6 to 12 months.

In certain embodiments, a therapeutic nucleic acid or compositionthereof is administered to a subject in combination with one or moreadditional therapies, such as a therapy described in Section 6.5.2. Thedosage of the other one or more additional therapies will depend uponvarious factors including, e.g., the therapy, the nature of theinfectious disease, the route of administration, the general health ofthe subject, etc. and should be decided according to the judgment of amedical practitioner. In specific embodiments, the dose of the othertherapy is the dose and/or frequency of administration of the therapyrecommended for the therapy for use as a single agent is used inaccordance with the methods disclosed herein. In other embodiments, thedose of the other therapy is a lower dose and/or less frequentadministration of the therapy than recommended for the therapy for useas a single agent is used in accordance with the methods disclosedherein. Recommended doses for approved therapies can be found in thePhysician's Desk Reference.

In certain embodiments, a therapeutic nucleic acid or compositionthereof is administered to a subject concurrently with theadministration of one or more additional therapies. In otherembodiments, a therapeutic nucleic acid or composition thereof isadministered to a subject every 3 to 7 days, 1 to 6 weeks, 1 to 5 weeks,1 to 4 weeks, 2 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks and one ormore additional therapies (such as described in Section 6.5.2) isadministered every 3 to 7 days, 1 to 6 weeks, 1 to 5 weeks, 1 to 4weeks, 1 to 3 weeks, or 1 to 2 weeks. In certain embodiments, atherapeutic nucleic acid or composition thereof is administered to asubject every 1 to 2 weeks and one or more additional therapies (such asdescribed in Section 6.5.2) is administered every 2 to 4 weeks. In someembodiments, a therapeutic nucleic acid or composition thereof isadministered to a subject every week and one or more additionaltherapies (such as described in Section 6.5.2) is administered every 2weeks.

7. EXAMPLES

The examples in this section (i.e., Section 7) are offered by way ofillustration, and not by way of limitation.

7.1 Example 1: mRNA Synthesis and Purification

DNA Linearization. DNA plasmid template containing the target sequenceencoding the 51 subunit, a few different versions of the receptorbinding domain (RBD), or the receptor binding motif (RBM) of thecoronavirus SARS-CoV-2 Spike (S) protein, the 5′ and 3′-UTR and polyAsignal was linearized using restriction enzyme digestion. Every 10 μg ofplasmid was mixed with 10 U of Esp3IBsmBI, incubated at 37° C. for 4hours to ensure complete linearization. The reaction was terminated byadding 1/10th volume of 3 M Na acetate (pH 5.5) and 2.5 volumes ofethanol, mix well and chill at −20° C. for 1 h. Linearized DNA wasprecipitated by centrifugation at 13800 g for 15 minutes at 4° C.,washed twice with 70% ethanol, resuspended in nuclease-free H₂O.

In vitro Transcription of mRNA. Contents of a typical 20 μL reactionmixture are shown in the table below:

Nuclease-free H₂O Up to 20 μL RNase Inhibitor(40 U/μL) 0.5 μL rNTPmixture (100 mM each) 8 μL (10 mM each final) 10X IVT Reaction Buffer 2μL 1M MgCl₂ 0.8 μL 0.1M DTT 2 μL 100 U/mL Pyrophosphatase Inorganic 0.8μL 100 mM NaCl 1 μL Linearized DNA 1 μg T7 RNA Polymerase (50 U/μL) 2 μL

The reaction mixture was incubated at 37° C. for 6 hours followed byaddition of 1p1 of DNase I (RNase-free, 1 U/μL) to remove the DNAtemplate, incubate for 30 minutes at 37° C. The synthesized RNA waspurified by adding 0.5 volume of 7.5 M LiCl 7.5 M LiCl, 50 mM EDTA andincubating at −20° C. for 45 minutes, followed by centrifugation at 4°C. for 15 minutes at 13800 g to pellet the mRNA. Then the supernatantwas removed and the pellet was rinsed twice with 500 μL of ice cold 70%ethanol, mRNA was resuspended in nuclease-free H₂O, adjustedconcentration to 1 mg/mL, and stored at −20° C.

mRNA Capping. Each 10 μg uncapped mRNA was heated at 65° C. for 10minutes, placed on ice for 5 minutes, and mixed with 10 U VacciniaCapping Enzyme, 50 U mRNA Cap 2′-O-Methyltransferase, 0.2 mM SAM, 0.5 mMGTP and 1U RNase inhibitor, and incubated at 37° C. for 60 minutes togenerate capl modification structure. The modified mRNA was precipitatedby LiCl as previously described and the RNA was resuspended innuclease-free H₂O, and stored at −20° C.

HPLC Purification. RNA was purified by high performance liquidchromatography (HPLC) using a C₄ column (5 μm) (10 mm×250 mm column).Buffer A contained 0.1 M triethylammonium acetate (TEAA), pH=7.0 andBuffer B contained 0.1 M TEAA, pH=7.0 and 25% acetonitrile.

FIG. 1 shows an exemplary HPLC purification of in vitro transcribedmRNA. As shown in FIG. 1 , mRNA molecules were successfully produced bythe in vitro transcription and maturation processes described above andwere purified from the reaction system using HPLC.

7.2 Example 2: In Vitro Transfection and Antigen Expression Analysis

Different mRNAs molecules encoding SARS-CoV-2 S protein antigensproduced in Example 1 were transfected into expression cell lines suchas HEK293F and Hela cultured cells to evaluate efficiency of in vitroexpression of the mRNA molecules.

To assemble the mRNA-lipid complex, two separate tubes were set up with1 μL of Lipofectamine mixed with 30 μL Opti-MEM, and with 1 μg mRNA with30 μL Opti-MEM, respectively. The two samples were mixed and incubatedat room temperature for 5 minutes. Fifty microliter of such a complexwas used to transfect cells present in 1 well of a 24-well plate, andthe cells were incubated in humidified 37° C./5% CO2 incubator untilanalysis.

Expression analysis. Cells were transferred from 24 hourspost-transfection culture, and centrifuged at 200 RCF for 5 minutes atroom temperature. Next, cells were treated with 4% (v/v)paraformaldehyde for 30 min, and washed with PBS. Next, cells weretreated with 0.2% (v/v) Triton X-100 for 10 min, and washed with PBS.Next, cells were blocked with 5% (w/v) bovine serum albumin for 1 h, andwashed with PBS. Next, cells were incubated with several rabbitanti-SARS-CoV-2 S protein antibodies at 4° C. for 1 h, and labeled withFITC-labeled anti-rabbit antibody (1:200) as secondary antibody for 30min, and washed with PBS and counter stained by DAPI. The signals wereexamined by confocal laser scanning microscopy.

Particularly, FIG. 2 shows exemplary confocal fluorescence microscopyimages of Hela cells transfected with an mRNA construct encoding aSARS-CoV-2 S protein RBD sequence (RBD sample 1). Cells were incubatedwith 3 different monoclonal antibodies recognizing the S protein RBD ofSARS-CoV-2, namely SARS-2-H014, SARS-2-mh001 and SARS-2-mh219,respectively.

As shown in FIG. 2 , in vitro transcribed mRNA molecules encoding theSARS-CoV-2 S protein RBD effectively transfected Hela cells. Thetransfected Hela cells expressed the encoded viral antigens at asatisfactory level, as can be recognized by the three monoclonalantibodies used in this study. The transfected Hela cells maintainednormal cellular morphology, indicating a lack of cellular toxicityresulted from the expression of the encoded viral antigen.

Western blot. For the secreted proteins such as SARS-CoV-2 S protein orits fragments, cultures of cells transfected with the mRNA moleculesproduced in Example 1 was collected and analyzed by Western blot 24hours post-transfection. Following SDS-PAGE, proteins were transferredonto blotting membrane. The blots were rinsed briefly with PBS and thenincubated with added a rabbit anti-Spike RBD antibody for 2 hr at RT.The blots were washed extensively in PBS. HRP-conjugated anti-rabbitantibody was added and incubated for 1 hr at RT with gentle agitation.The membrane was washed with PBS, and incubated with added appropriateenzyme substrate solution to visualize protein bands.

FIG. 3 shows an exemplary Western blot analysis of culture supernatantof Hela cells transfected with an mRNA molecule encoding a SARS-CoV-2 Sprotein RBD sequence. Particularly, the lanes labeled “RBD sample 1” and“RBD sample 2” were loaded with culture supernatant of Hela cellstransfected with mRNA constructs encoding different SARS-CoV-2 S proteinRBD sequences described herein, respectively. The lane labeled“rRBD-His” was loaded a recombinantly produced SARS-CoV-2 S protein RBDsequence fused to a C-terminal His-tag. The lane labeled “NT” was loadedcell culture supernatant of Hela cells transfected with an irrelevantmRNA construct as a negative control group.

As shown in FIG. 3 , the in vitro transcribed mRNA constructs encodingthe SARS-CoV-2 S protein RBD effectively transfected Hela cells. Thetransfected Hela cells expressed and secreted the encoded viral antigenat a satisfactory level. The bands around about 30kD corresponded tosecreted viral antigen in the monomeric form. The bands around about60kD corresponded to secreted viral antigen in the dimeric form. Withoutbeing bound by the theory, it is contemplated that multimerized forms ofsecreted viral antigen can be more immunogenic and effective in inducinghumoral immune response upon administration to a vaccinated subject ascompared to monomeric forms. As shown in FIG. 3 , the viral antigenencoded by the mRNA construct can multimerize after expression,indicating usefulness of the mRNA construct in eliciting an immuneresponse against the virus upon administration to a subject.

ELISA. The quantity of mRNA-encoded viral peptide or protein expressedin cell culture supernatant was determined by ELISA. Particularly, toperform the ELISA assays, microtiter plate wells was coated with 100 μlsolution containing 5 μg/ml SARS-CoV-2 S Protein RBD, and incubated at4° C. for 12 hours with closure plate membrane. Next, the plate waswashed 3 times in washing buffer. Next, 300 μl of 5% BSA in PBST wasadded to each well and incubated for 60 minutes at 37° C. Next, theplate was washed 4 times in washing buffer. Next, culture supernatantsamples and SARS-CoV-2 S protein RBD standards were diluted in washingbuffer and 100 μl of suitably diluted samples and standards were addedto the relevant wells in triplicates. Next, the wells were incubated for60 minutes at 37° C., and washed 3 times in washing buffer. Next, 100 μLof rabbit anti-SARS-CoV-2 S protein antibody was added to each well ofplate. Next, the plate was covered and incubated for 60 minutes at 37°C., and washed 3 times in washing buffer. Next, 100 μl of dilutedHRP-conjugated anti-Rabbit antibody was added to each well, andincubated for 1 hour at 37° C., and washed 3 times in washing buffer.Next, 100 μl of TMB substrate solution was added to each well, incubatedat room temperature (and in the dark if required) for approximately 10min. Next, 100 μL of Stop Solution was added to each well, and gentlyand thoroughly mixed. Next, a Molecular Devices plate reader was used toread ODs at 450/620 nm subtracted for detection.

FIG. 4 shows an exemplary ELISA analysis measuring proteinconcentrations (ng/mL) of mRNA-encoded SARS-CoV-2 S protein RBD inculture supernatant of cells transfected with two mRNA constructs,designated as “RBD sample 1” and “RBD sample 2” respectively. BSA wasused as a negative control for ELISA. This study further demonstratedthat cells transfected with the mRNA constructs expressed and secretedthe encoded viral antigen at satisfactory levels, as quantified byELISA.

7.3 Example 3. Production of Neutralizing Antibodies by Mice Vaccinatedwith mRNA Containing LNP

BALB/c mice were vaccinated by intramuscular injection of 100 μL of LNPformulation containing 10 μg of rRNA encoding a SARS-CoV-2 S protein RBD(RBD sample 1), and blood was collected from tail veins on day 7, 14, 21and 28 post-vaccination, respectively. A group of vaccinated mice werealso boosted by receiving a second intramuscular injection of the samedose of LNP formulation containing the mRNA 14 days after the firstinjection, and blood was collected from tail veins on day 7, 14, 21 and28 after the second boosting injection. The 50% plaque reductionneutralization titer (PRNT 50) values of the collected mouse serum weredetermined to evaluate the production of neutralizing antibody by thevaccinated animals.

PRNT assays. To perform the plaque reduction neutralization titer (PRNT)assays, the serum sample or solution of antibody to be tested wasdiluted and mixed with a viral suspension. The mixture was thenincubated to allow the antibody to react with the virus. Next, themixture was poured over a confluent monolayer of host cells. The surfaceof the cell layer was covered in a layer of agarose or carboxymethylcellulose to prevent the virus from spreading indiscriminately. Theconcentration of plaque forming units (PFU) can be estimated by thenumber of plaques (regions of infected cells) formed a few days later.Depending on the virus, the plaque forming units can be measured bymicroscopic observation, fluorescent antibodies or specific dyes thatreact with infected cells. The concentration of serum to reduce thenumber of plaques by 50% compared to the serum free virus gives themeasure of how much antibody is present or how effective it is. Thismeasurement is denoted as the PRNT 50 value.

Particularly, in this study, mouse serum collected as described abovewere heat inactivated at 55° C. for 30 min and then serially diluted to1:50, 1:100, 1:200, 1:400, and 1:800 in PBS. Equal volume of PBScontaining 100 PFU of SARS-CoV-2 pseudovirus was added to each serumdilution. Each mixture was incubated at 37° C. for 30 min, added toconfluent cultures of Vero E6 monolayers, and allowed them to incubateat 37° C. for 60 minutes. The cell monolayers were covered with 4 ml of0.8% agarose melted in standard Vero E6 cell medium, and plaques wereresolved the with neutral red staining 2 days later. The PRNT 50 valueswere then calculated and plotted in FIG. 5 . Particularly, Y axis showsthe reciprocal of PRNT 50 values (i.e., 1/PRNT 50). X-axis shows thegroup of animals as follows: “RBD” denotes mice receiving only the firstinjection and the “RBD-B” denotes mice receiving the first and theboosting injection. “Control” denotes a group of mice receivedintramuscular injection of 100 μl LNP formulation without mRNA andboosted with the same dose of blank LNP 14 days later.

As shown in FIG. 5 , animals vaccinated with the therapeuticmRNA-containing LNP produced neutralizing antibodies that significantlyreduced infection of cells by SARS-CoV-2. This study demonstrated thatthe present therapeutic mRNA-containing LNP composition can be used fortreating, managing or preventing infection by the coronavirusSARS-CoV-2.

7.4 Example 4: Study of Correlation Between RBD Expression in Mice andmRNA Contents of mRNA-LNP Samples

The following experiment was performed to establish a method fordetecting RBD expression in animals in response to a finishedcoronavirus SARS-CoV-2 mRNA vaccine product, so as to explore thecorrelation between the RBD expression and the mRNA contents in thefinished product.

Animal grouping and dosing information were as follows:

Dosing Number concen- Dosing Dosing Adminis- Group Experimental oftration volume amount tration No. groups animals (μg/mL) (μL) (μg) route1 Blank control 10 — — — — group 2 Vaccine sample 4 10 100 1 iv., once.group E 3 Vaccine sample 4 20 100 2 iv., once. group D 4 Vaccine sample4 30 100 3 iv., once. group C 5 Vaccine sample 4 40 100 4 iv., once.group B 6 Vaccine sample 4 50 100 5 iv., once. group A

Experimental animals, ICH mice, were administrated with different testsubstances by intravenous injection according to the table above. 6hours after the administration, blood was collected from the heart afterdeep carbon dioxide anesthesia. After standing at room temperature forabout 30 min, the serum was separated (4° C., 8000 rpm (5724 g), 10min), sub packaged (in triplicate, each larger than 110 ul; if it wasnot able to guarantee that all of the three aliquots met therequirement, at least two of them would do, and the blood volume pertube was marked), and stored at −80° C. in a refrigerator. Serum sampleswere collected from the remaining 10 blank mice, mixed well and thendispensed into small tubes.

The RBD expression in the serum samples was detected by ELISA method asfollows:

All the samples and reagents were recovered to room temperature beforeuse.

-   -   1) 100 μL of ACE2 coating stock solution was added to the ELISA        plate, and the plate was sealed with a sealing membrane, and        incubated at 4° C. for 15 hours.    -   2) The liquid in the wells was discarded, and the plate was        washed with a washing buffer for 3 times, 300 μL/well, and        soaked for 2 min each time.    -   3) 300 μL/well of blocking solution was added, and the plate was        sealed with a sealing membrane, and incubated at 37° C. for 1        hour.    -   4) The liquid in the wells was discarded, and the plate was        washed with a washing buffer for 3 times, 300 μL/well, and        soaked for 2 min each time.    -   5) 100 μL of diluted samples to be tested and a standard were        added to the ELISA plate, and the plate was sealed with a        sealing membrane, and incubated at 37° C. for 1 hour.    -   6) The liquid in the wells was discarded, and the plate was        washed with a washing buffer for 3 times, 300 μL/well, and        soaked for 2 min each time.    -   7) 100 μL of primary antibody stock solution was added to each        well of the plate, and the plate was covered with a sealing        membrane, and incubated at 37° C. for 1 hour. 8) The liquid in        the wells was discarded, and the plate was washed with a washing        buffer for 3 times, 300 μL/well, and soaked for 2 min each time.    -   9) 100 μL of HRP secondary antibody stock solution was added to        each well of the plate, and the plate was covered with a sealing        membrane, and incubated at 37° C. for 1 hour.    -   10) The liquid in the wells was discarded, and the plate was        washed with a washing buffer for 3 times, 300 μL/well, and        soaked for 2 min each time.    -   11) 100 μL of TMB substrate solution was added to each        microwell, and the plate was incubated at room temperature,        protected from the light, for about 5 min.    -   12) 100 μL of ELISA stop solution was added to each microwell.    -   13) The result was determined by using a multifunctional        microplate reader, where the detection wavelengths were set as        450 nm and 620 nm, a Curve Fit/5-parameter regression model was        set for the Standard Curve, and the Dilution Factor of the test        samples was set as 2, while standard curve and sample settings        being performed (the template settings can be set before the        start of the reading), and where the absorption values were        determined.    -   14) The result was calculated automatically via a software by        using a 5-parameter regression model of the protein        concentration (X) of the protein standard and its corresponding        fluorescence value (Y), so as to calculate and obtain the        protein concentrations (Unk-dilution/AdjResult) in the samples.

The detection results of the RBD contents in the serum samples of theexperimental animals were shown in FIG. 6 and Table 6. Among all mouseserum samples, no RBD concentration was detected in the “blank serum”sample. It was observed that there was a significant dose-dependencebetween 1 ug and 5 ug mRNA, and the expressed RBD contents were0.14-2.18 ng/mL.

TABLE 6 Result Dilu- AdjResult Average Sample Value (ng/mL) tion (ng/mL)(ng/mL) Blank control group-1 0.101 ND 2 ND ND Blank control group-20.130 ND 2 ND Blank control group-3 0.118 ND 2 ND Blank control group-40.131 ND 2 ND Vaccine sample group E-1 0.163 0.079 2 0.158 0.14 Vaccinesample group E-2 0.146 0.022 2 0.044 Vaccine sample group E-3 0.1760.109 2 0.218 Vaccine sample group E-4 0.157 0.063 2 0.126 Vaccinesample group D-1 0.15  0.038 2 0.076 0.29 Vaccine sample group D-2 0.2370.217 2 0.434 Vaccine sample group D-3 0.308 0.321 2 0.642 Vaccinesample group D-4 0.145 0.014 2 0.027 Vaccine sample group C-1 0.44 0.501 2 1.002 0.83 Vaccine sample group C-2 0.372 0.409 2 0.818 Vaccinesample group C-3 0.405 0.454 2 0.908 Vaccine sample group C-4 0.2860.29  2 0.579 Vaccine sample group B-1 0.453 0.519 2 1.038 1.21 Vaccinesample group B-2 0.464 0.534 2 1.067 Vaccine sample group B-3 0.6110.743 2 1.486 Vaccine sample group B-4 0.524 0.618 2 1.236 Vaccinesample group A-1 1.029 1.499 2 2.998 2.18 Vaccine sample group A-2 0.6910.865 2 1.73  Vaccine sample group A-3 0.715 0.902 2 1.805 Vaccinesample group A-4 0.825 1.089 2 2.178

7.5 Example 5: Antigen Immunogenicity Analysis

The purpose of the following experiment was to evaluate theimmunogenicity of the mRNA molecular liposome (RBD mRNA-LNP) loaded withthe SARS-CoV-2 coronavirus S-RBD protein of the present invention.

The number of animals in each group and the detailed immunizationroutes, doses and schedules were shown in the table below. Experimentalanimals, BALB/c mice, received the test antigen (10 Ng/50 μL per mouse)via a single point intramuscular injection on the right hind limb on day0. A same dose of the test vaccine was vaccinated again on day 14. Thedetailed administration methods, dosing amounts and administrationroutes were as follows:

Number Immuni- of Injection zation Immunity Group animals Test antigenDose volume method cycle^(a) 1 8 RBD 10 μg per 50 μL i.m. Days 0mRNA-LNP animal per and 14 animal 2 4 Empty 10 μg per 50 μL i.m. Days 0liposome animal per and 14 animal 3 4 1 × PBS N/A 50 μL i.m. Days 0 perand 14 animal Note: ^(a)The day of the first immunization was defined asday 0.

Before the first immunization, 4 mice were randomly selected to collectblood to prepare serum samples (above 150 μL), and the serum sampleswere collected without anticoagulants for monitoring, as shown in thefollowing table.

Time Group point Sample collection and processing method RBD Day 14Collecting whole blood to prepare serum samples mRNA- before the secondimmunization, serum amount LNP >150 μL per animal group Day 21Collecting whole blood to prepare serum samples, serum amount >150 μLper animal Day 28 Collecting whole blood to prepare serum samples, serumamount >150 μL per animal, and Collecting spleens from 4 animals toprepare a single cell suspension Empty Day 14 Collecting whole blood toprepare serum samples liposome before the second immunization, serumamount group >150 μL per animal Day 21 Collecting whole blood to prepareserum samples, serum amount >150 μL per animal Day 28 Collecting wholeblood to prepare serum samples, serum amount >150 μL per animal, andCollecting spleen from 1 animal to prepare a single cell suspension 1 ×PBS Day 14 Collecting whole blood to prepare serum samples group beforethe second immunization, serum amount >150 μL per animal Day 21Collecting whole blood to prepare serum samples, serum amount >150 μLper animal Day 28 Collecting whole blood to prepare serum samples, serumamount >150 μL per animal, and Collecting spleen from 1 animal toprepare a single cell suspension

There were 52 samples in total as follows: 4 serum samples collectedbefore the first immunization; 16 serum samples collected 14 days afterthe first immunization; 16 serum samples collected 21 days after thefirst immunization; and 16 serum samples collected 28 days after thefirst immunization, respectively. After the serum collection wascompleted, the coronavirus RBD IgG titers were detected together. Inthis experiment, a “Mouse Anti-New Coronavirus (2019-nCoV)S-RBD ProteinIgG Antibody Detection Kit” developed by Wantai BioPharm was used forIgG titer detection. The test serum samples were diluted in a 10-foldgradient starting from 1:10 with a sample diluent, and shaken gently tomix well. 100 μL of each of the diluted samples, the negative controland the positive control was added to each well. The plate was sealedwith a sealing membrane. The sealing membrane was cut off at 37° C. for30 min, and the plate was washed for 5 times, 300 μL each time, anddried at the last time. 100 μL of ELISA reagent was added to each well,except for blank wells. The sealing membrane was cut off at 37° C. for30 min, and the plate was washed for 5 times, 300 μL each time. 50 mL ofeach of color developer A and B was added to each well, shaken gently tomix well, and developed at 37° C., protected from the light, for 15 min.50 μL of the stop solution was added to each well, and mixed well in agentle manner. The result was measured within 10 min. The wavelength ofthe microplate reader was set at 450 nm. The maximum dilution factor forwhich the detection result was positive was selected, and Titer resultwas the OD value of the positive maximum dilutionfactor/0.1*corresponding dilution factor.

Specifically, the mice were immunized with a single dose (10 μg) of themRNA vaccine on day 0, and a booster dose (10 μg) was given on day 14.The anti-S-RBD IgG antibody levels in the mouse serum samples weredetected 14, 21, and 29 days after the immunization. The results wereshown in FIG. 7 . In the group of mice vaccinated with mRNA-LNP, oneweek after the second immunization, the specific IgG titer increasedfrom about 1/900 on day 14 to 1/70,000 on day 21 and maintained at thesame level on day 29. In contrast, neither of the empty liposome and PBScontrol groups had RBD-specific IgG expression. This result clearlyshowed that the vaccine product described in the present invention hadstrong immunogenicity and can specifically induce the production ofrelated antibodies to achieve the effect of controlling or preventingthe infection of the coronavirus SARS-CoV-2.

8. SEQUENCE LISTING

The present specification is being filed with a computer readable form(CRF) copy of the Sequence Listing. The CRF entitled 14639-009-228SequenceListing.txt, which was created on Apr. 2, 2021 and is 199,381bytes in size, and is incorporated herein by reference in its entirety.

1.-83. (canceled)
 84. A nucleic acid encoding a polypeptide derived fromthe spike (S) protein of coronavirus SARS-COV-2, wherein the polypeptideis an immunogenic fragment of the S protein comprising the amino acidsequence of SEQ ID NO:16, wherein the immunogenic fragment of the Sprotein does not contain the full-length S protein.
 85. The nucleic acidof claim 84, wherein the immunogenic fragment of the S protein comprisesthe receptor binding motif (RBM) of the S protein.
 86. The nucleic acidof claim 84, wherein the immunogenic fragment of the S protein comprisesthe receptor binding motif (RBM) of the S protein and a S proteinmutation corresponding to the N501T amino acid substitution.
 87. Thenucleic acid of claim 84, wherein the nucleic acid is an mRNA molecule.88. A composition comprising the nucleic acid of claim 84 and at leastone lipid.
 89. The composition of claim 88, wherein the at least onelipid forms a lipid nanoparticle encapsulating the nucleic acid.
 90. Thecomposition of claim 89, wherein the nucleic acid is an mRNA.
 91. Thecomposition of claim 90, wherein the composition is a pharmaceuticalcomposition.
 92. The composition of claim 91, wherein the pharmaceuticalcomposition is a vaccine.
 93. A method for eliciting an immune responseto coronavirus SARS-COV-2 in a system, wherein the method comprisescontacting the composition of claim 88 with the system under a suitablecondition that allows expression of the nucleic acid in the system. 94.The method of claim 93, wherein the system is a subject, and wherein thecontacting is performed by administration to the subject.
 95. The methodof claim 93, wherein the immune response comprises production of aneutralizing antibody against coronavirus SARS-COV-2.
 96. A method forreducing viral titer of coronavirus SARS-COV-2 in a system, wherein themethod comprises contacting the composition of claim 88 with the systemunder a suitable condition that allows expression of the nucleic acid inthe system.
 97. The method of claim 96, wherein the system is a subject,and wherein the contacting is performed by administration to thesubject.